Microorganisms and methods for producing sialylated and n-acetylglucosamine-containing oligosaccharides

ABSTRACT

The invention provides compositions and methods for engineering bacteria to produce sialylated and N-acetylglucosamine-containing oligosaccharides, and the use thereof in the prevention or treatment of infection.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/776,216, filed on Sep. 14, 2015, which is a national stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2014/029804, filed on Mar. 14, 2014, which claims benefit of, and priority to, U.S. Provisional Application No. 61/782,999, filed on Mar. 14, 2013; the contents of each of which are hereby incorporated by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “37847_512001 WO_ST25.txt”, which was created on Sep. 11, 2017, and is 144 kilobytes in size, are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention provides compositions and methods for producing purified oligosaccharides, in particular certain N-acetylglucosamine-containing and/or sialylated oligosaccharides that are typically found in human milk.

BACKGROUND OF THE INVENTION

Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides (human milk oligosaccharides, hMOS). Many of these molecules are not utilized directly by infants for nutrition, but they nevertheless serve critical roles in the establishment of a healthy gut microbiome, in the prevention of disease, and in immune function. Prior to the invention described herein, the ability to produce hMOS inexpensively at large scale was problematic. For example, hMOS production through chemical synthesis was limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost. As such, there is a pressing need for new strategies to inexpensively manufacture large quantities of hMOS for a variety of commercial applications.

SUMMARY OF THE INVENTION

The invention described herein features efficient and economical methods for producing N-acetylglucosamine-containing and/or sialylated oligosaccharides.

The invention provides a method for producing an N-acetylglucosamine-containing oligosaccharide in a bacterium comprising the following steps: providing a bacterium that comprises an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and a functional lactose permease; and culturing the bacterium in the presence of lactose. The N-acetylglucosamine-containing oligosaccharide is then retrieved from the bacterium or from a culture supernatant of the bacterium.

The invention further provides a method for producing a sialylated oligosaccharide in a bacterium comprising the following steps: providing a bacterium that comprises an exogenous sialyl-transferase gene, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, and a functional lactose permease gene; and culturing the bacterium in the presence of lactose. The sialylated oligosaccharide is then retrieved from the bacterium or from a culture supernatant of the bacterium. Specifically, a sialic acid synthetic capability comprises expressing exogenous CMP-Neu5Ac synthetase, an exogenous sialic acid synthase, and an exogenous UDP-GlcNAc-2-epimerase, or a functional variant or fragment thereof.

In both methods for producing N-acetylglucosamine-containing and/or sialylated oligosaccharides, it is preferable that the bacterium further comprises the capability for increased UDP-GlcNAc production. By “increased production capability” is meant that the host bacterium produces greater than 10%, 20%, 50%, 100%, 2-fold, 5-fold, 10-fold, or more of a product than the native, endogenous bacterium. Preferably, the bacterium over-expresses a positive endogenous regulator of UDP-GlcNAc synthesis. For example, the bacterium overexpresses the nagC gene of Escherichia coli. Alternatively, the bacterium over-expresses the Escherichia coli glmS (L-glutamine:D-fructose-6-phosphate aminotransferase) gene, or alternatively, over-expresses the Escherichia coli glmY gene (a positive translational regulator of glmS), or, alternatively over-expresses the Escherichia coli glmZ gene (another positive translational regulator of glmS: glmY and glmZ are described in Reichenbach et al Nucleic Acids Res 36, 2570-80 (2008)). Alternatively, the bacterium over-expresses any combination of such approaches. For example, the bacterium over-expresses nagC and glmS. Alternatively, the bacterium over-expresses nagC and glmY. Alternatively, the bacterium over-expresses nagC and glmZ. The methods also further encompass over-expressing any functional variant or fragment of nagC, glmS, glmY and glmZ and any combination thereof. By “overexpression” is meant that the gene transcript or encoded gene product is 10%, 20%, 50%, 2-fold, 5-fold, 10-fold, or more than the level expressed or produced by the corresponding native, naturally-occurring, or endogenous gene.

The invention described herein details the manipulation of genes and pathways within bacteria such as the enterobacterium Escherichia coli K12 (E. coli) leading to high level synthesis of hMOS. Other strains of E. coli for suitable for use in the present invention include E. coli MG1655, E. coli W3110, E. coli DH5aE, E. coli B, E. coli C, and E. coli W. A variety of bacterial species are suitable for use in the oligosaccharide biosynthesis methods, for example Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus are suitable for use, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus are modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bacteroides (e.g., Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides fine goldii, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides xylanisolvens), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Parabacteroides (e.g. Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides johnsonii and Parabacteroides merdae), Prevotella (e.g., Prevotella copri), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and an N-acetylglucosamine-containing or sialylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The N-acetylglucosamine-containing or sialylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products.

The bacterium comprises a deleted or inactivated (i.e., non-functional) endogenous β-galactosidase gene. For example, the β-galactosidase gene comprises an E. coli lacZ gene (e.g., GenBank Accession Number V00296.1 (GI:41901), incorporated herein by reference). The endogenous lacZ gene of the E. coli is deleted or functionally inactivated, but in such a way that expression of the downstream lactose permease (lacY) gene remains intact, i.e. a functional lactose permease gene is also present in the bacterium. By deleted is meant that a portion or the whole coding sequence is absent, such that no gene product is produced. An “inactivated” gene does not produce a gene product that functions as the native, naturally-occurring, or endogenous gene. For example, the functional activity of an inactivated β-galactosidase gene product is reduced to 10%, 20%, 50%, or 100%, 1-fold, 2-fold, 5-fold, or 10-fold less than the functional activity of the native, naturally-occurring, endogenous gene product.

The lactose permease gene is an endogenous lactose permease gene or an exogenous lactose permease gene. For example, the lactose permease gene comprises an E. coli lacY gene (e.g., GenBank Accession Number V00295.1 (GI:41897), incorporated herein by reference). Many bacteria possess the inherent ability to transport lactose from the growth medium into the cell, by utilizing a transport protein that is either a homolog of the E. coli lactose permease (e.g., as found in Bacillus licheniformis), or a transporter that is a member of the ubiquitous PTS sugar transport family (e.g., as found in Lactobacillus casei and Lactobacillus rhamnosus). For bacteria lacking an inherent ability to transport extracellular lactose into the cell cytoplasm, this ability is conferred by an exogenous lactose transporter gene (e.g., E. coli lacY) provided on recombinant DNA constructs, and supplied either on a plasmid expression vector or as exogenous genes integrated into the host chromosome.

For the production of N-acetylglucosamine-containing oligosaccharides, the bacterium comprises an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene or a functional variant or fragment thereof. This exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene is obtained from any one of a number of sources, e.g., the LgtA gene described from N. meningitides (SEQ ID NO:16 Genbank protein Accession AAF42258.1, incorporated herein by reference) or N. gonorrhoeae (Genbank protein Accession ACF31229.1). Optionally, an additional exogenous glycosyltransferase gene is co-expressed in the bacterium comprising an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase. For example, a β-1,4-galactosyltransferase gene is co-expressed with the UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene. This exogenous β-1,4-galactosyltransferase gene is obtained from any one of a number of sources, e.g., that described from N. meningitidis, the LgtB gene (Genbank protein Accession AAF42257.1), or from H. pylori, the Lex2B gene (SEQ ID NO:17 Genbank protein Accession NP_207619.1, incorporated herein by reference). Optionally, the additional exogenous glycosyltransferase gene co-expressed in the bacterium comprising an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene is a ⊖-1,3-galactosyltransferase gene, e.g., that described from E. coli O55:H7, the WbgO gene (SEQ ID NO:18 Genbank protein Accession YP_003500090.1, incorporated herein by reference), or from H. pylori, the jhp0563 gene (Genbank protein Accession AEZ55696.1). Functional variants and fragments of any of the enzymes described above are also encompassed by the present invention.

In one embodiment, the N-aceteylglucosamine-containing oligosaccharides produced by the methods described herein include Lacto-N-triose 2 (LNT2), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lacto-N-fucopentaose V (LNF V), Lacto-N-difucohexaose I (LDFH I), Lacto-N-difucohexaose II (LDFH II), and Lacto-N-neodifucohexaose II (LFNnDFH II).

For the production of sialyl-oligosaccharides, the bacterium comprises an exogenous sialyl-transferase gene. For example, the exogenous sialyl-transferase gene encodes α(2,3) sialyl-transferase or the exogenous sialyl-transferase gene encodes α(2,6) sialyl-transferase or the exogenous sialyl-transferase gene encodes α(2,8) sialyltransferase. The exogenous sialyl-transferase genes is obtained from any one of a number of sources, e.g., those described from N. meningitidis, N. gonorrhoeae, and from a number of organisms of the genus Photobacterium. Examples of α(2,8) sialyltransferases, useful for the production of polysialic acid for example, are found in Campylobacter jejuni (CstII: ADN52706) and Neisseria meningitides (or siaD:AAA20478).

The bacteria used herein to produce hMOS are genetically engineered to comprise an increased intracellular lactose pool (as compared to wild type) and to comprise UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and/or sialyl-transferase activity. Optionally, they also comprise β-1,4-galactosyltransferase or β-1,3-galactosyltransferase activity, and/or α-1,2-, α-1,3- and/or α-1,4-fucosyltransferase activity. In some cases, the bacterium further comprises a functional, wild-type E. coli lacZ⁺ gene inserted into an endogenous gene, for example, the ion gene in E. coli or the thyA gene in E. coli. In this manner, the bacterium further comprises a mutation in a ion gene or a mutation in the thyA gene. In these cases, the endogenous lacZ gene of the E. coli is deleted or functionally inactivated, but in such a way that expression of the downstream lactose permease (lacY) gene remains intact. The organism so manipulated maintains the ability to transport lactose from the growth medium, and to develop an intracellular lactose pool for use as an acceptor sugar in oligosaccharide synthesis, while also maintaining a low level of intracellular beta-galactosidase activity useful for a variety of additional purposes. For example, the invention also includes: a) methods for phenotypic marking of a gene locus in a β-galactosidase negative host cell by utilizing a β-galactosidase (e.g., lacZ) gene insert engineered to produce a low but readily detectable level of β-galactosidase activity, b) methods for readily detecting lytic bacteriophage contamination in fermentation runs through release and detection of cytoplasmic β-galactosidase in the cell culture medium, and c) methods for depleting a bacterial culture of residual lactose at the end of production runs. a), b) and c) are each achieved by utilizing a functional β-galactosidase (e.g., lacZ) gene insert carefully engineered to direct the expression of a low, but detectable level of β-galactosidase activity in an otherwise β-galactosidase negative host cell. The bacterium optionally further comprises a mutation in a lacA gene. Preferably, the bacterium accumulates an increased intracellular lactose pool, and produces a low level of beta-galactosidase. An increased intracellular pool is wherein the concentration of lactose in the host bacterium at least 10%, 20%, 50%, 2-fold, 5-fold, or 10-fold higher than that of the native, naturally-occurring bacterium.

In one aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding an UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and an exogenous nucleic acid encoding β-1,4-galactosyltransferase is lacto-N-neotetraose (LNnT). In another aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding a UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and an exogenous nucleic acid encoding β-1,3-galactosyltransferase is lacto-N-tetraose (LNT).

Described herein are compositions comprising a bacterial cell that produces the human milk oligosaccharide LNnT (lacto-N-neotetraose), wherein the bacterial cell comprises an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and an exogenous nucleic acid encoding a β-1,4-galactosyltransferase. Preferably, the bacterial cell is E. coli. The exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene is obtained from any one of a number of sources, e.g., the LgtA gene described from N. meningitides. The exogenous β-1,4-galactosyltransferase gene is obtained from any one of a number of sources, e.g., that described from N. meningitidis, the LgtB gene, or from H. pylori, the jhp0765 gene.

Additionally, the bacterium preferably comprises increased production of UDP-GlcNAc. An exemplary means to achieve this is by over-expression of a positive endogenous regulator of UDP-GlcNAc synthesis, for example, overexpression of the nagC gene of Escherichia coli. In one aspect, this nagC over-expression is achieved by providing additional copies of the nagC gene on a plasmid vector or by integrating additional nagC gene copies into the host cell chromosome. Alternatively, over-expression is achieved by modulating the strength of the ribosome binding sequence directing nagC translation or by modulating the strength of the promoter directing nagC transcription. As further alternatives the intracellular UDP-GlcNAc pool may be enhanced by other means, for example by over-expressing the Escherichia coli glmS (L-glutamine:D-fructose-6-phosphate aminotransferase) gene, or alternatively by over-expressing the Escherichia coli glmY gene (a positive translational regulator of glmS), or alternatively by over-expressing the Escherichia coli glmZ gene (another positive translational regulator of glmS), or alternatively by simultaneously using a combination of approaches. In one preferred embodiment, for example, the nagC (SEQ ID NO:19 Genbank protein Accession BAA35319.1, incorporated herein by reference) and glmS (SEQ ID NO:20 Genbank protein Accession NP_418185.1, incorporated herein by reference) genes which encode the sequences provided herein are overexpressed simultaneously in the same host cell in order to increase the intracellular pool of UDP-GlcNAc. Other components of UDP-GlcNAc metabolism include: (GlcNAc-1-P)N-acetylglucosamine-1-phosphate; (GlcN-1-P) glucosamine-1-phosphate; (GlcN-6-P) glucosamine-6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate. Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and lacto-N-neotetraose is retrieved, either from the bacterium itself (i.e., by lysis) or from a culture supernatant of the bacterium.

Also within the invention is an isolated E. coli bacterium as described above and characterized as comprising a deleted or inactivated endogenous β-galactosidase gene, an inactivated or deleted lacA gene, and a functional lactose permease (lacY) gene.

Also described herein are compositions comprising a bacterial cell that produces the human milk oligosaccharide 6′-SL (6′-sialyllactose), wherein the bacterial cell comprises an exogenous sialyl-transferase gene encoding α(2,6)sialyl-transferase. Preferably, the bacterial cell is E. coli. The exogenous sialyl-transferase gene utilized for 6′-SL production is obtained from any one of a number of sources, e.g., those described from a number of organisms of the genus Photobacterium. In yet another aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding an α(2,3) sialyltransferase is 3′-SL (3′-sialyllactose). The exogenous sialyltransferase gene utilized for 3′-SL production is obtained from any one of a number of sources, e.g., those described from N. meningitidis and N. gonorrhoeae.

Additionally, the bacterium contains a deficient sialic acid catabolic pathway. By “sialic acid catabolic pathway” is meant a sequence of reactions, usually controlled and catalyzed by enzymes, which results in the degradation of sialic acid. An exemplary sialic acid catabolic pathway in Escherichia coli is described herein. In the sialic acid catabolic pathway described herein, sialic acid (NeuSAc; N-acetylneuraminic acid) is degraded by the enzymes NanA (N-acetylneuraminic acid lyase) and NanK (N-acetylmannosamine kinase) and NanE (N-acetylmannosamine-6-phosphate epimerase), all encoded in the nanATEK-yhcH operon, and repressed by NanR (ecocyc.org/ECOLI). A deficient sialic acid catabolic pathway is engineered in Escherichia coli by way of a mutation in endogenous nanA (N-acetylneuraminate lyase) (e.g., GenBank Accession Number D00067.1 (GI:216588), incorporated herein by reference) and/or nanK (N-acetylmannosamine kinase) genes (e.g., GenBank Accession Number (amino acid) BAE77265.1 (GI:85676015), incorporated herein by reference), and/or nanE (N-acetylmannosamine-6-phosphate epimerase, GI: 947745, incorporated herein by reference). Optionally, the nanT (N-acetylneuraminate transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P)N-acetylmannosamine-6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate. In some preferred embodiments, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated, while nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, while nanK has not been mutated, inactivated or deleted. A mutation is one or more changes in the nucleic acid sequence coding the gene product of nanA, nanK, nanE, and/or nanT. For example, the mutation may be 1, 2, 5, 10, 25, 50 or 100 changes in the nucleic acid sequence. For example, the nanA, nanK, nanE, and/or nanT is mutated by a null mutation. Null mutations as described herein encompass amino acid substitutions, additions, deletions, or insertions that either cause a loss of function of the enzyme (i.e., reduced or no activity) or loss of the enzyme (i.e., no gene product). By deleted is meant that the coding region is removed in whole or in part such that no gene product is produced. By inactivated is meant that the coding sequence has been altered such that the resulting gene product is functionally inactive or encodes a gene product with less than 100%, 80%, 50%, or 20% of the activity of the native, naturally-occurring, endogenous gene product. A “not mutated” gene or protein does not differ from a native, naturally-occurring, or endogenous coding sequence by 1, 2, 5, 10, 20, 50, 100, 200 or 500 more codons, or to the corresponding encoded amino acid sequence.

Moreover, the bacterium (e.g., E. coli) also comprises a sialic acid synthetic capability. For example, the bacterium comprises a sialic acid synthetic capability through provision of an exogenous UDP-GlcNAc 2-epimerase (e.g., neuC of Campylobacter jejuni (SEQ ID NO: 13, GenBank AAK91727.1; GI:15193223, incorporated herein by reference) or equivalent (e.g. E. coli S88 neuC GenBank YP_002392936.1; GI: 218560023), a NeuSAc synthase (e.g., neuB of C. jejuni (SEQ ID NO:14 AAK91726.1GenBank GI:15193222, incorporated herein by reference) or equivalent, (e.g. Flavobacterium limnosediminis sialic acid synthase, GenBank GI:559220424), and/or a CMP-Neu5Ac synthetase (e.g., neuA of C. jejuni (SEQ ID NO: 15 GenBank AAK91728.1; GI:15193224, incorporated herein by reference) or equivalent, (e.g. Vibrio brasiliensis CMP-sialic acid synthase, GenBank GI: 493937153). Functional variants and fragments are also disclosed herein.

Additionally, the bacterium comprising a sialic acid synthetic capability preferably increased production of UDP-GlcNAc. An exemplary means to achieve this is by over-expression of a positive endogenous regulator of UDP-GlcNAc synthesis, for example, simultaneous overexpression of the nagC and glmS genes of Escherichia coli. This nagC and glmS over-expression is achieved by providing additional copies of the nagC and glmS genes on a plasmid vector, or by integrating additional nagC and glmS gene copies into the host cell chromosome. Alternatively, over-expression is achieved by modulating the strength of the ribosome binding sequence directing nagC (described by Sleight et al, Nucleic Acids Res. May 2010; 38(8): 2624-2636) and/or glmS translation, or by modulating the strength of the promoter/s directing nagC and glmS transcription (Sleight et al, Nucleic Acids Res. May 2010; 38(8): 2624-2636)

Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and, in the instance where cells comprise an α(2,6) sialyltransferase (e.g. Photobacterium spp JT-ISH-224 (SEQ ID NO:21 Genbank protein Accession BAF92026.1, incorporated herein by reference), 6′-sialyllactose is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. In the instance where cells comprise an α(2,3) sialyltransferase, (e.g. Neisseria meningitidis 1st (Genbank protein Accession NP273962.1) 3′-sialyllactose is recovered either from the bacterium itself (e.g., by lysis of the bacterium) or from a culture supernatant of the bacterium.

Also within the invention is an isolated E. coli bacterium as described above and characterized as comprising a deleted or inactivated endogenous β-galactosidase gene, an exogenous sialyl-transferase gene, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, a deleted lacA gene, and a functional lactose permease (lacY) gene.

A purified N-acetylglucosamine-containing or sialylated oligosaccharide produced by the methods described above is also within the invention. A purified oligosaccharide, e.g., 6′-SL, is one that is at least 90%, 95%, 98%, 99%, or 100% (w/w) of the desired oligosaccharide by weight. Purity is assessed by any known method, e.g., thin layer chromatography or other electrophoretic or chromatographic techniques known in the art. The invention includes a method of purifying an N-acetylglucosamine-containing or sialylated oligosaccharide produced by the genetically engineered bacteria described above, which method comprises separating the desired N-acetylglucosamine-containing or sialylated oligosaccharide (e.g., 6′-SL) from contaminants in a bacterial cell extract or lysate, or bacterial cell culture supernatant. Contaminants include bacterial DNA, protein and cell wall components, and yellow/brown sugar caramels sometimes formed in spontaneous chemical reactions in the culture medium.

The oligosaccharides are purified and used in a number of products for consumption by humans as well as animals, such as companion animals (dogs, cats) as well as livestock (bovine, equine, ovine, caprine, or porcine animals, as well as poultry). For example, a pharmaceutical composition comprising purified 6′-sialyllactose (6′-SL) and an excipient is suitable for oral administration. Large quantities of 6′-SL are produced in bacterial hosts, e.g., an E. coli bacterium comprising a heterologous sialyltransferase, e.g., a heterologous α(2,6)sialyltransferase. An E. coli bacterium comprising an enhanced cytoplasmic pool of each of the following: lactose and CMP-Neu5Ac, is useful in such production systems. In the case of lactose, endogenous E. coli metabolic pathways and genes are manipulated in ways that result in the generation of increased cytoplasmic concentrations of lactose, as compared to levels found in wild type E. coli. For example, the bacteria contain at least 10%, 20%, 50%, 2×, 5×, 10× or more of the levels in a corresponding wild type bacteria that lacks the genetic modifications described above. In the case of CMP-NeuSAc, endogenous NeuSAc catabolism genes are inactivated and exogenous CMP-NeuSAc biosynthesis genes introduced into E. coli resulting in the generation of a cytoplasmic pool of CMP-NeuSAc not found in the wild type bacterium.

A method of producing a pharmaceutical composition comprising a purified hMOS is carried out by culturing the bacterium described above, purifying the hMOS produced by the bacterium, and combining the hMOS with an excipient or carrier to yield a dietary supplement for oral administration. These compositions are useful in methods of preventing or treating enteric and/or respiratory diseases in infants and adults. Accordingly, the compositions are administered to a subject suffering from or at risk of developing such a disease using known methods of clinical therapy.

The invention also provides for increasing, in E. coli, the intracellular concentration of the nucleotide sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). This is achieved by over-expressing the bi-functional endogenous positive regulator of UDP-GlcNac synthesis and repressor of glucosamine and N-acetylglucosamine catabolism, nagC, simultaneously with the gene encoding L-glutamine:D-fructose-6-phosphate aminotransferase, glmS.

The invention also provides for increasing the intracellular concentration of lactose in E. coli, for cells grown in the presence of lactose, by using manipulations of endogenous E. coli genes involved in lactose import, export, and catabolism. In particular, described herein are methods of increasing intracellular lactose levels in E. coli genetically engineered to produce a human milk oligosaccharide by incorporating a lacA mutation into the genetically modified E. coli. The lacA mutation prevents the formation of intracellular acetyl-lactose, which not only removes this molecule as a contaminant from subsequent purifications, but also eliminates E. coli's ability to export excess lactose from its cytoplasm, thus greatly facilitating purposeful manipulations of the E. coli intracellular lactose pool.

Also described herein are bacterial host cells with the ability to accumulate a intracellular lactose pool while simultaneously possessing low, functional levels of cytoplasmic β-galactosidase activity, for example as provided by the introduction of a functional recombinant E. coli lacZ gene, or by a β-galactosidase gene from any of a number of other organisms (e.g., the lac4 gene of Kluyveromyces lactis (e.g., GenBank Accession Number M84410.1 (GI:173304), incorporated herein by reference). Low, functional levels of cytoplasmic β-galactosidase include β-galactosidase activity levels of between 0.05 and 200 units, e.g., between 0.05 and 5 units, between 0.05 and 4 units, between 0.05 and 3 units, or between 0.05 and 2 units (for standard definition see: Miller J H, Laboratory CSH. Experiments in molecular genetics. Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.; 1972; incorporated herein by reference). This low level of cytoplasmic β-galactosidase activity, while not high enough to significantly diminish the intracellular lactose pool, is nevertheless very useful for tasks such as phenotypic marking of desirable genetic loci during construction of host cell backgrounds, for detection of cell lysis due to undesired bacteriophage contaminations in fermentation processes, for the facile removal of undesired residual lactose at the end of fermentations, or for in-process fermentation QC purposes (i.e. as a non-standard phenotype the provision of a weak lacZ phenotype aids in culture purity assessments).

Methods of purifying a N-acetylglucosamine-containing or sialylated oligosaccharide produced by the methods described herein are carried out by binding the oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of the bacterium to a carbon column, and subsequently eluting it from the column. Purified N-acetylglucosamine-containing or sialylated oligosaccharides are produced by the methods described herein.

Optionally, the invention features a vector, e.g., a vector containing a nucleic acid. The vector can further include one or more regulatory elements, e.g., a heterologous promoter. The regulatory elements can be operably linked to a protein gene, fusion protein gene, or a series of genes linked in an operon in order to express the fusion protein. To maintain the plasmid vector stably within the cell a selectable marker is included within its sequence, such as an antibiotic resistance gene or a gene that complements a nutritional auxotrophy of the host bacterium. For example, in E. coli, a thymidine deficiency caused by a chromosomal defect in the thymidylate synthase gene (thyA) can be complemented by a plasmid borne wild type copy of the thyA (M. Belfort, G. F. Maley, F. Maley, Proceedings of the National Academy of Sciences 80, 1858 (1983)) gene. Alternatively an adenine deficiency caused by a chromosomal deficiency in the adenylosuccinate synthetase (purA) gene (S. A. Wolfe, J. M. Smith, J Biol Chem 263, 19147-53 (1988)) can be complemented by a plasmid borne wild type copy of purA. Two plasmid vectors may be utilized simultaneously within the same bacterial cell by employing separate selectable markers, for example one plasmid utilizing thyA selection and one utilizing purA selection, and by utilizing two compatible plasmid replicons, for example in E. coli two such compatible replicons comprise the ColE1 (pUC) replicon and the p15A (pACYC) replicon (R. E. Bird, J Bacteriol 145, 1305-9 (1981)). In yet another aspect, the invention comprises an isolated recombinant cell, e.g., a bacterial cell containing aforementioned nucleic acid molecule/s or vector/s. The nucleic acid sequences can be optionally integrated into the genome.

The invention provides a method of treating, preventing, or reducing the risk of infection in a subject comprising administering to said subject a composition comprising a human milk oligosaccharide, purified from a culture of a recombinant strain of the current invention, wherein the hMOS binds to a pathogen and wherein the subject is infected with or at risk of infection with the pathogen. In one aspect, the infection is caused by a Norwalk-like virus or Campylobacter jejuni. The subject is preferably a mammal in need of such treatment. The mammal is, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig. In a preferred embodiment, the mammal is a human. For example, the compositions are formulated into animal feed (e.g., pellets, kibble, mash) or animal food supplements for companion animals, e.g., dogs or cats, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably, the purified hMOS is formulated into a powder (e.g., infant formula powder or adult nutritional supplement powder, each of which is mixed with a liquid such as water or juice prior to consumption) or in the form of tablets, capsules or pastes or is incorporated as a component in dairy products such as milk, cream, cheese, yogurt or kefir, or as a component in any beverage, or combined in a preparation containing live microbial cultures intended to serve as probiotics, or in prebiotic preparations intended to enhance the growth of beneficial microorganisms either in vitro or in vivo. For example, the purified sugar (e.g., LNnT or 6′-SL) can be mixed with a Bifidobacterium or Lactobacillus in a probiotic nutritional composition. (i.e. Bifidobacteria are beneficial components of a normal human gut flora and are also known to utilize hMOS for growth.

All genes described herein also include a description of the corresponding encoded gene products. As such, the uses of exogenous genes as described herein encompass nucleic acids that encode the gene product sequences disclosed herein. The person skilled in the art could readily generate nucleic acid sequences that encode the protein sequences described herein and introduce such sequences into expression vectors to carry out the present invention.

The term “substantially pure” in reference to a given polypeptide, polynucleotide or oligosaccharide means that the polypeptide, polynucleotide or oligosaccharide is substantially free from other biological macromolecules. The substantially pure polypeptide, polynucleotide or oligosaccharide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate calibrated standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, thin layer chromatography (TLC) or HPLC analysis.

Polynucleotides, polypeptides, and oligosaccharides of the invention are purified and/or isolated. Purified defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. Specifically, as used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or oligosaccharide, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. For example, purified hMOS compositions are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate calibrated standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, thin layer chromatography (TLC) or HPLC analysis. For example, a “purified protein” refers to a protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. Preferably, the protein constitutes at least 10, 20, 50 70, 80, 90, 95, 99-100% by dry weight of the purified preparation.

By “isolated nucleic acid” is meant a nucleic acid that is free of the genes that flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide.

A “heterologous promoter”, when operably linked to a nucleic acid sequence, refers to a promoter which is not naturally associated with the nucleic acid sequence.

The term “over-express” as used herein refers to gene transcript or encoded gene product is 10%, 20%, 50%, 2-fold, 5-fold, 10-fold, or more than the level expressed or produced by a native, naturally-occurring, or endogenous gene in a bacterium in which it naturally occurs. For example, the host bacterium described herein are engineered to over-express an exogenous gene transcript or encoded gene product of UDP-GlcNAc:Galα/β-R 3-N-acetylglucosaminyltransferase, nagC, glmS, glmY, glmZ, a sialyl-transferase, a β-galactosyltransferase, an α-fucosyltransferase, CMP-Neu5Ac synthetase, a sialic acid synthase, or a UDP-GlcNAc 2-epimerase, i.e., a gene or gene product with a sequence corresponding to that of a bacterium other than the host bacterium.

The terms “treating” and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage. The terms “preventing” and “prevention” refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.

By the terms “effective amount” and “therapeutically effective amount” of a formulation or formulation component is meant a nontoxic but sufficient amount of the formulation or component to provide the desired effect.

The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer 2′-fucosyllactose (2′-FL) synthesis in Escherichia coli (E. coli). Specifically, the lactose synthesis pathway and the GDP-fucose synthesis pathway are illustrated. In the GDP-fucose synthesis pathway: manA=phosphomannose isomerase (PMI), manB=phosphomannomutase (PMM), manC=mannose-1-phosphate guanylyltransferase (GMP), gmd=GDP-mannose-4,6-dehydratase,fcl=GDP-fucose synthase (GFS), and ΔwcaJ=mutated UDP-glucose lipid carrier transferase.

FIG. 2 is a schematic demonstrating metabolic pathways involved in the synthesis of UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) and catabolism of glucosamine and N-acetylglucosamine in E. coli. In the schematic: (GlcNAc-1-P)N-acetylglucosamine-1-phosphate; (GlcN-1-P) glucosamine-1-phosphate; (GlcN-6-P) glucosamine-6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate; glmS (L-glutamine:D-fructose-6-phosphate aminotransferase), glmM (phosphoglucosamine mutase), glmU (fused N-acetyl glucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyl transferase), nagC (bifunctional transcriptional activator/repressor protein), nagA (N-acetylglucosamine-6-phosphate deacetylase) and nagB (glucosamine-6-phosphate deaminase), nagE (N-acetylglucosamine transporter] and manXYZ [glucosamine transporter).

FIG. 3 is a schematic demonstrating metabolic pathways and one example (utilizing nanT, nanA and nanK deletions) of the changes introduced into them to engineer 6′-sialyllactose (6′-SL) synthesis in E. coli. Abbreviations include: (NeuSAc)N-acetylneuraminic acid, sialic acid; (AnanT) mutated N-acetylneuraminic acid transporter; (ΔnanA) mutated N-acetylneuraminic acid lyase; (ManNAc)N-acetylmannosamine; (ΔnanK) mutated N-acetylmannosamine kinase; (nanE) wild-type N-acetylmannosamine-6-phosphate epimerase; (ManNAc-6-P)N-acetylmannosamine-6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; (Fruc-6-P) Fructose-6-phosphate; (neuA), CMP-N-acetylneuraminic acid synthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; (neuB), N-acetylneuraminic acid synthase; (neuC) UDP-GlcNAc-2-epimerase; and (UDP-GlcNAc) uridine diphosphate N-acetylglucosamine.

FIG. 4 is a schematic that illustrates the new configuration of genes engineered at the Escherichia coli thyA locus in strains used to produce N-acetylglucosamine-containing oligosaccharides.

FIG. 5 is a plasmid map of pG292, which expresses the N. meningitidis β(1,3)-N-acetylglucosaminyltransferase gene lgtA.

FIG. 6 is a plasmid map of pG221, which expresses, as an operon, the N. meningitidis β(1,3)-N-acetylglucosaminyltransferase gene lgtA and the E. coli O55:H7 wbgO β(1,3)-galactosyltransferase gene.

FIG. 7 is a plasmid map of pG222, which expresses, as an operon, the N. meningitidis β(1,3)-N-acetylglucosaminyltransferase gene lgtA and the H. pylori 4GalT (jhp0765) β(1,4)-galactosyltransferase gene.

FIG. 8 illustrates schematically the enzymatic reactions necessary to produce from lactose, via the intermediate trisaccharide lacto-N-triose 2 (LNT2), the two human milk oligosaccharides: Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT). A thin layer chromatogram (on left) is presented of culture medium samples taken from small scale E. coli cultures and demonstrating synthesis of LNT2, LNT and LNnT. A second thin layer chromatogram (on right) is presented of culture medium samples taken from a 15 L E. coli bioreactor culture—demonstrating synthesis of LNnT.

FIG. 9 is a plasmid map of pG317, a low-copy vector which expresses as an operon, under the control of the E. coli lac promoter, the Campylobacter jejuni ATCC43438 neuB, neuC and neuA genes, encoding N-acetylneuraminate synthase, UDP-N-acetylglucosamine 2-epimerase, and N-acetylneuraminate cytidylyltransferase, respectively.

FIG. 10 is a plasmid map of pG315, a multi-copy vector which expresses a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224, under the control of the E. coli lac promoter.

FIG. 11 is a photograph of a thin layer chromatogram showing 6′-SL in culture medium produced by E. coli strain E547 (ΔnanRATEK), containing plasmids expressing a bacterial α(2,3)sialyltransferase and neuA, neuB and neuC. FIG. 11 also shows a TLC analysis of culture supernatants from two fermentations producing 6′-sialylactose (6′-SL). Samples to the left of the figure are taken from a fermentation of an E. coli strain containing pG315 (carrying a strong RBS in front of the α(2,6)sialyltransferase gene in the vector). Samples on the right of the figure are taken from a fermentation of an E. coli strain containing a close variant of pG315 that carries a weaker RBS in front of the α(2,6)sialyltransferase gene.

FIG. 12 is a plasmid map of pG345, a multi-copy vector which expresses a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224, under the control of a weaker ribosomal binding site (SEQ ID NO:8) and the E. coli lac promoter.

FIG. 13 is a schematic demonstrating metabolic pathways and a second example (utilizing nanT, nanA and nanE deletions) of the changes introduced into them to engineer 6′-sialyllactose (6′-SL) synthesis in E. coli. Abbreviations include: (NeuSAc)N-acetylneuraminic acid, sialic acid; (AnanT) mutated N-acetylneuraminic acid transporter; (ΔnanA) mutated N-acetylneuraminic acid lyase; (ManNAc)N-acetylmannosamine; (nanK) wild-type N-acetylmannosamine kinase; (ΔnanE) mutated N-acetylmannosamine-6-phosphate epimerase; (ManNAc-6-P)N-acetylmannosamine-6-phosphate; (GlcNAc-6-P)N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; (Fruc-6-P) Fructose-6-phosphate; (neuA), CMP-N-acetylneuraminic acid synthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; (neuB), N-acetylneuraminic acid synthase; (neuC) UDP-GlcNAc-2-epimerase; and (UDP-GlcNAc) uridine diphosphate N-acetylglucosamine.

FIG. 14 illustrates the TLC analysis of cell pellets and or supernatants from a three pilot scale fermentation experiments using three E. coli strains carrying various combinations of nan mutations

FIG. 15 is a schematic illustrating the location of the gene deletion made within the E. coli nan operon to generate the [nanR+, nanA, nanT, nanE, nanK+] mutant locus of strains E1017 and E1018.

FIG. 16 is a cell density growth curve plot of four cultures of E680 transformed with pG292, induced or un-induced by tryptophan addition, and in the presence or absence of lactose in the growth medium. Abundant cell lysis is seen in the lactose-containing cultures.

FIG. 17 is a plasmid map of pG356, which expresses, as an operon, the E. coli glmS and nagC genes. pG356 carries a p15A replication origin and both ampC and purA selectable markers.

FIG. 18 is a fementation parameter trace and TLC culture supernatant analysis (for LNnT production) of a 1.5 L bioreactor culture of E796 transformed with pG222.

FIG. 19 is a fementation parameter trace and TLC culture supernatant analysis (for LNnT production) of a 1.5 L bioreactor culture of E866 transformed with both pG222 and pG356.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are genetic constructs and methods for production of N-acetylglucosamine-containing human milk oligosaccharides (hMOS) and sialyloligosaccharides. In order to make both N-acetylglucosamine-containing and sialyl-containing hMOS, one needs to tap into the cellular UDP-GlcNAc pool. Doing so can be challenging, since UDP-GlcNAc is an essential metabolite for bacteria (used to make the cell wall). The constructs, compositions, and methods of the invention overcome difficulties of the past by enhancing the UDP-GlcNAc pool, a strategy that represents an advantage in the production of both classes of hMOS. Other distinctions over earlier approaches represent improvements and/or confer advantages over those earlier strategies.

hMOS

Human milk glycans, which comprise both oligosaccharides (hMOS) and their glycoconjugates, play significant roles in the protection and development of human infants, and in particular the infant gastrointestinal (GI) tract. Milk oligosaccharides found in various mammals differ greatly, and their composition in humans is unique (Hamosh M., 2001 Pediatr Clin North Am, 48:69-86; Newburg D. S., 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milk change throughout lactation and also vary widely among individuals (Morrow A. L. et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Previously, a full exploration of the roles of hMOS was limited by the inability to adequately characterize and measure these compounds. In recent years sensitive and reproducible quantitative methods for the analysis of both neutral and acidic hMOS have been developed (Erney, R., Hilty, M., Pickering, L., Ruiz-Palacios, G., and Prieto, P. (2001) Adv Exp Med Biol 501, 285-297. Bao, Y., and Newburg, D. S. (2008) Electrophoresis 29, 2508-2515). Approximately 200 distinct oligosaccharides have been identified in human milk, and combinations of a small number of simple epitopes are responsible for this diversity (Newburg D. S., 1999 Curr Med Chem, 6:117-127; Ninonuevo M. et al., 2006 J Agric Food Chem, 54:7471-74801). hMOS are composed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid, Neu5Ac, NANA). hMOS are usually divided into two groups according to their chemical structures: neutral compounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, and acidic compounds including the same sugars, and often the same core structures, plus NANA (Charlwood J. et al., 1999 Anal Biochem, 273:261-277; Martin-Sosa et al., 2003 J Dairy Sci, 86:52-59; Parkkinen J. and Finne J., 1987 Methods Enzymol, 138:289-300; Shen Z. et al., 2001 J Chromatogr A, 921:315-321). Approximately 70-80% of oligosaccharides in human milk are fucosylated. A smaller proportion of the oligosaccharides in human milk are sialylated, or are both fucosylated and sialylated.

Interestingly, hMOS as a class, survive transit through the intestine of infants very efficiently, a function of their being poorly transported across the gut wall and of their resistance to digestion by human gut enzymes (Chaturvedi, P., Warren, C. D., Buescher, C. R., Pickering, L. K. & Newburg, D. S. Adv Exp Med Biol 501, 315-323 (2001)). One consequence of this survival in the gut is that hMOS are able to function as prebiotics, i.e. they are available to serve as an abundant carbon source for the growth of resident gut commensal microorganisms (Ward, R. E., Niñonuevo, M., Mills, D. A., Lebrilla, C. B., and German, J. B. (2007) Mol Nutr Food Res 51, 1398-1405). Recently, there is burgeoning interest in the role of diet and dietary prebiotic agents in determining the composition of the gut microflora, and in understanding the linkage between the gut microflora and human health (Roberfroid, M., Gibson, G. R., Hoyles, L., McCartney, A. L., Rastall, R., Rowland, I., Wolvers, D., Watzl, B., Szajewska, H., Stahl, B., Guarner, F., Respondek, F., Whelan, K., Coxam, V., Davicco, M. J., Léotoing, L., Wittrant, Y., Delzenne, N. M., Cani, P. D., Neyrinck, A. M., and Meheust, A. (2010) Br J Nutr 104 Suppl 2, S1-63).

A number of human milk glycans possess structural homology to cell receptors for enteropathogens, and serve roles in pathogen defense by acting as molecular receptor “decoys”. For example, pathogenic strains of Campylobacter bind specifically to glycans in human milk containing the H-2 epitope, i.e., 2′-fucosyl-N-acetyllactosamine or 2′-fucosyllactose (2′-FL); Campylobacter binding and infectivity are inhibited by 2′-FL and other glycans containing this H-2 epitope (Ruiz-Palacios, G. M., Cervantes, L. E., Ramos, P., Chavez-Munguia, B., and Newburg, D. S. (2003) J Biol Chem 278, 14112-14120). Similarly, some diarrheagenic E. coli pathogens are strongly inhibited in vivo by hMOS containing 2′-linked fucose moieties. Several major strains of human caliciviruses, especially the noroviruses, also bind to 2′-linked fucosylated glycans, and this binding is inhibited by human milk 2′-linked fucosylated glycans. Consumption of human milk that has high levels of these 2′-linked fucosyloligosaccharides has been associated with lower risk of norovirus, Campylobacter, ST of E. coli-associated diarrhea, and moderate-to-severe diarrhea of all causes in a Mexican cohort of breastfeeding children (Newburg D. S. et al., 2004 Glycobiology, 14:253-263; Newburg D. S. et al., 1998 Lancet, 351:1160-1164). Several pathogens are also known to utilize sialylated glycans as their host receptors, such as influenza (Couceiro, J. N., Paulson, J. C. & Baum, L. G. Virus Res 29, 155-165 (1993)), parainfluenza (Amonsen, M., Smith, D. F., Cummings, R. D. & Air, G. M. J Virol 81, 8341-8345 (2007), and rotoviruses (Kuhlenschmidt, T. B., Hanafin, W. P., Gelberg, H. B. & Kuhlenschmidt, M. S. Adv Exp Med Biol 473, 309-317 (1999)). The sialyl-Lewis X epitope is used by Helicobacter pylori (Mandavi, J., Sondén, B., Hurtig, M., Olfat, F. O., et al. Science 297, 573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P., Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and some strains of noroviruses (Rydell, G. E., Nilsson, J., Rodriguez-Diaz, J., Ruvoën-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).

The nucleotide sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a key metabolic intermediate in bacteria, where it is involved in the synthesis and maintenance of the cell envelope. In all known bacterial classes, UDP-GlcNAc is used to make peptidoglycan (murein); a polymer comprising the bacterial cell wall whose structural integrity is absolutely essential for growth and survival. In addition, gram-negative bacteria use UDP-GlcNAc for the synthesis of lipid A, an important component of the outer cell membrane. Thus, for bacteria, the ability to maintain an adequate intracellular pool of UDP-GlcNAc is critical.

Biosynthesis of certain human milk oligosaccharides (hMOS) has been achieved in engineered strains of the bacterium, Escherichia coli K12. As described herein, simple fucosylated hMOS, e.g. 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), and lactodifucotetraose (LDFT), are produced efficiently by live E. coli through artificially enhancing existing intracellular pools of GDP-fucose (the nucleotide sugar donor) and lactose (the accepting sugar), and by then using these enhanced pools as substrates for heterologous recombinant fucosyltransferases (FIG. 1). Since neither the lactose nor GDP-fucose pools are essential for E. coli survival, biosynthesis of simple fucosylated hMOS is achieved at good yields without negative consequences on the host bacterium's growth or viability. However, to synthesize more complex hMOS in E. coli, use of the critical bacterial UDP-GlcNAc pool is required, with consequent potential impacts on cell viability.

The UDP-GlcNAc pool in E. coli is produced through the combined action of three glm genes, glmS (L-glutamine:D-fructose-6-phosphate aminotransferase), glmM (phosphoglucosamine mutase), and the bifunctional glmU (fused N-acetyl glucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyl transferase) (FIG. 2). These three genes direct a steady flow of carbon to UDP-GlcNAc, a flow that originates with fructose-6-phosphate (an abundant molecule of central energy metabolism). Expression of the glm genes is under positive control by the transcriptional activator protein, NagC.

When E. coli encounters glucosamine or N-acetyl-glucosamine in its environment, these molecules are each transported into the cell via specific membrane transport proteins and are used either to supplement the flow of carbon to the UDP-GlcNAc pool, or alternatively they are consumed to generate energy, under the action of nag operon gene products (i.e. nagA [N-acetylglucosamine-6-phosphate deacetylase] and nagB [glucosamine-6-phosphate deaminase]). In contrast to the glm genes, expression of nagA and nagB are under negative transcriptional control, but by the same regulatory protein as the glm genes, i.e. NagC. NagC is thus bi-functional, able to activate UDP-GlcNAc synthesis, while at the same time repressing the degradation of glucosamine-6-phosphate and N-acetylglucosamine-6-phosphate.

The binding of NagC to specific regulatory DNA sequences (operators), whether such binding results in gene activation or repression, is sensitive to fluctuations in the cytoplasmic level of the small-molecule inducer and metabolite, GlcNAc-6-phosphate. Intracellular concentrations of GlcNAc-6-phosphate increase when N-acetylglucosamine is available as a carbon source in the environment, and thus under these conditions the expression of the glm genes (essential to maintain the vital UDP-GlcNAc pool) would decrease, unless a compensatory mechanism is brought into play. E. coli maintains a baseline level of UDP-GlcNAc synthesis through continuous expression of nagC directed by two constitutive promoters, located within the upstream nagA gene. This constitutive level of nagC expression is supplemented approximately threefold under conditions where the degradative nag operon is induced, and by this means E. coli ensures an adequate level of glm gene expression under all conditions, even when N-acetylglucosamine is being utilized as a carbon source.

Many hMOS incorporate GlcNAc into their structures directly, and many also incorporate sialic acid, a sugar whose synthesis involves consumption of UDP-GlcNAc (FIG. 3, FIG. 13). Thus, synthesis of many types of hMOS in engineered E. coli carries the significant risk of reduced product yield and compromised cell viability resulting from depletion of the bacterium's UDP-GlcNAc pool. One way to address this problem during engineered synthesis of GlcNAc- or sialic acid-containing hMOS is to boost the UDP-GlcNAc pool through simultaneous over-expression of nagC, or preferably by simultaneous over-expression of both nagC and glmS.

While studies suggest that human milk glycans could be used as prebiotics and as antimicrobial anti-adhesion agents, the difficulty and expense of producing adequate quantities of these agents of a quality suitable for human consumption has limited their full-scale testing and perceived utility. What has been needed is a suitable method for producing the appropriate glycans in sufficient quantities at reasonable cost. Prior to the invention described herein, there were attempts to use several distinct synthetic approaches for glycan synthesis. Novel chemical approaches can synthesize oligosaccharides (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003)), but reactants for these methods are expensive and potentially toxic (Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). Enzymes expressed from engineered organisms (Albermann, C., Piepersberg, W. & Wehmeier, U. F. Carbohydr Res 334, 97-103 (2001); Bettler, E., Samain, E., Chazalet, V., Bosso, C., et al. Glycoconj J 16, 205-212 (1999); Johnson, K. F. Glycoconj J 16, 141-146 (1999); Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, E. J. Curr Opin Chem Biol 4, 110-119 (2000)) provide a precise and efficient synthesis (Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999)); Crout, D. H. & Vic, G. Curr Opin Chem Biol 2, 98-111 (1998)), but the high cost of the reactants, especially the sugar nucleotides, limits their utility for low-cost, large-scale production. Microbes have been genetically engineered to express the glycosyltransferases needed to synthesize oligosaccharides from the bacteria's innate pool of nucleotide sugars (Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S., Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000); Endo, T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316, 179-183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. Nat Biotechnol 16, 847-850 (1998)). However, low overall product yields and high process complexity have limited the commercial utility of these approaches.

Prior to the invention described herein, which enables the inexpensive production of large quantities of neutral and acidic hMOS, it had not been possible to fully investigate the ability of this class of molecule to inhibit pathogen binding, or indeed to explore their full range of potential additional functions.

Prior to the invention described herein, chemical syntheses of hMOS were possible, but were limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003); Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). Also, prior to the invention described herein, in vitro enzymatic syntheses were also possible, but were limited by a requirement for expensive nucleotide-sugar precursors. The invention overcomes the shortcomings of these previous attempts by providing new strategies to inexpensively manufacture large quantities of human milk oligosaccharides for use as dietary supplements. The invention described herein makes use of an engineered bacterium E. coli (or other bacteria) engineered to produce sialylated oligosaccharides in commercially viable levels, for example the methods described herein enable the production of 3′-SL at >50 g/L in bioreactors.

Variants and Functional Fragments

The present invention features introducing exogenous genes into bacterium to manipulate the pathways to increase UDP-GlcNAc pools, to produce sialylated oligosaccharides and to produce N-acetylglucosamine-containing oligosaccharides. In any of the methods described herein, the genes or gene products may be variants or functional fragments thereof.

A variant of any of genes or gene products disclosed herein may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein. The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof.

Variants as disclosed herein also include homolog, orthologs, or paralogs of the genes or gene products described herein that retain the same biological function as the genes or gene products specified herein. These variants can be used interchangeably with the genes recited in these methods. Such variants may demonstrate a percentage of homology or identity, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, preferably in a functional domain, e.g. catalytic domain.

For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402). For the PSI-BLAST search, the following exemplary parameters are employed: (1) Expect threshold was 10; (2) Gap cost was Existence: 11 and Extension: 1; (3) The Matrix employed was BLOSUM62; (4) The filter for low complexity regions was “on”.

Changes can be introduced by mutation into the nucleic acid sequence or amino acid sequence of any of the genes or gene products described herein, leading to changes in the amino acid sequence of the encoded protein or enzyme, without altering the functional ability of the protein or enzyme. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of any of sequences expressly disclosed herein. A “non-essential” amino acid residue is a residue at a position in the sequence that can be altered from the wild-type sequence of the polypeptide without altering the biological activity, whereas an “essential” amino acid residue is a residue at a position that is required for biological activity. For example, amino acid residues that are conserved among members of a family of proteins are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are poorly conserved among members of the protein family) may not be as essential for activity and thus are more likely to be amenable to alteration. Thus, another aspect of the invention pertains to nucleic acid molecules encoding the proteins or enzymes disclosed herein that contain changes in amino acid residues relative to the amino acid sequences disclosed herein that are not essential for activity.

An isolated nucleic acid molecule encoding a protein homologous to any of the genes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.

Mutations can be introduced into a nucleic acid sequence such that the encoded amino acid sequence is altered by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for mediating oligossacharide modification, synthesis, or degradation (via detection of the products).

The present invention also provides for functional fragments of the genes or gene products described herein. A fragment, in the case of these sequences and all others provided herein, is defined as a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.

For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 350 amino acids, 350 to 400 amino acids, 400 to 450 amino acids, or 450 to 500 amino acids. The fragments encompassed in the present invention comprise fragments that retain functional fragments. As such, the fragments preferably retain the catalytic domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. The biological function of said fragment can be measured by measuring ability to synthesize or modify a substrate oligosaccharide, or conversely, to catabolize an oligosaccharide substrate.

Example 1: Engineering of E. coli to Generate Host Strains for the Production of N-Acetylglucosamine-Containing Human Milk Oligosaccharides

The E. coli K12 prototroph, W3110, was chosen as the parent background for hMOS biosynthesis. This strain had previously been modified at the ampC locus by the introduction of a tryptophan-inducible P_(trpB)-cI+ repressor construct (McCoy, J. & Lavallie, E. Current protocols in molecular biology/edited by Frederick M. Ausubel et al., (2001)), enabling economical production of recombinant proteins from the phage λ P_(L) promoter (Sanger, F., Coulson, A. R., Hong, G. F., Hill, D. F. & Petersen, G. B. J Mol Biol 162, 729-773 (1982)) through induction with millimolar concentrations of tryptophan (Mieschendahl, M., Petri, T. & Hänggi, U. Nature Biotechnology 4, 802-808 (1986)). The strain GI724, an E. coli W3110 derivative containing the tryptophan-inducible P_(trpB)-cI+ repressor construct in ampC, was used at the basis for further E. coli strain manipulations

Biosynthesis of hMOS requires the generation of an enhanced cellular pool of lactose. This enhancement was achieved in strain GI724 through several manipulations of the chromosome using λ Red recombineering (Court, D. L., Sawitzke, J. A. & Thomason, L. C. Annu Rev Genet 36, 361-388 (2002)) and generalized P1 phage transduction (Thomason, L. C., Costantino, N. & Court, D. L. Mol Biol Chapter 1, Unit 1.17 (2007)). The ability of the E. coli host strain to accumulate intracellular lactose was first engineered by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lacI). During construction of this deletion, the lacIq promoter was placed immediately upstream of the lactose permease gene, lacY. The modified strain thus maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type copy of the lacZ (β-galactosidase) gene responsible for lactose catabolism. An intracellular lactose pool is therefore created when the modified strain is cultured in the presence of exogenous lactose.

An additional modification useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of hMOS) is the incorporation of a lacA mutation. LacA is a lactose acetyltransferase that is only active when high levels of lactose accumulate in the E. coli cytoplasm. High intracellular osmolarity (e.g., caused by a high intracellular lactose pool) can inhibit bacterial growth, and E. coli has evolved a mechanism for protecting itself from high intra cellular osmolarity caused by lactose by “tagging” excess intracellular lactose with an acetyl group using LacA, and then actively expelling the acetyl-lactose from the cell (Danchin, A. Bioessays 31, 769-773 (2009)). Production of acetyl-lactose in E. coli engineered to produce human milk oligosaccharides is therefore undesirable: it reduces overall yield. Moreover, acetyl-lactose is a side product that complicates oligosaccharide purification schemes. The incorporation of a lacA mutation resolves these problems, as carrying a deletion of the lacA gene renders the bacterium incapable of synthesizing acetyl-lactose.

A thyA (thymidylate synthase) mutation was introduced by almost entirely deleting the thyA gene and replacing it by an inserted functional, wild-type, but promoter-less E. coli lacZ⁺ gene carrying the 2.8 ribosome binding site (SEQ ID NO: 10) (ΔthyA::(2.8RBS lacZ⁺,kan^(r)). λ Red recombineering was used to perform the construction. FIG. 4 illustrates the new configuration of genes thus engineered at the thyA locus. The complete DNA sequence of the region, with annotations in GenBank format is disclosed herein. Genomic DNA sequence surrounding the lacZ+ insertion into the thyA region is set forth in SEQ ID NO: 1.

The thyA defect can be complemented in trans by supplying a wild-type thyA gene on a multicopy plasmid (Belfort, M., Maley, G. F. & Maley, F. Proceedings of the National Academy of Sciences 80, 1858 (1983)). This complementation is used herein as a means of plasmid maintenance (eliminating the need for a more conventional antibiotic selection scheme to maintain plasmid copy number).

The genotype of strain E680 is given below. E680 incorporates all the changes discussed above and is a host strain suitable for the production of N-acetylglucosamine-containing oligosaccharides.

F′402 proA+B+, PlacIq-lacY, Δ(lacI-lacZ) 158, ΔlacA398/araC, Δgpt-mhpC, ΔthyA::(2.8RBS lacZ+, KAN), rpoS+, rph+, ampC::(Ptrp T7g10 RBS-λcl+, CAT)

E796 is a strain similar to E680 and carries a thyA (thymidylate synthase) mutation, introduced by almost entirely deleting the thyA gene and replacing it by an inserted functional, wild-type, but promoter-less E. coli lacZ⁺ gene but carrying the 0.8 ribosome binding site (SEQ ID NO: 11) [ΔthyA::(0.8RBS lacZ+, KAN)]. The genotype of strain E796 is given below. E796 incorporates all the changes discussed above and is a host strain suitable for the production of N-acetylglucosamine-containing oligosaccharides.

F′402 proA+B+, PlacIq-/acY, Δ(lacI-lacZ) 158, ΔlacA398/araC, Δgpt-mhpC, ΔthyA::(2.8RBS lacZ+, KAN), rpoS+, rph+, ampC::(Ptrp T7g10 RBS-λcl+, CAT)

E866 is a strain similar to E796 and is useful for dual plasmid selection. E866 also carries a thyA (thymidylate synthase) mutation, introduced by almost entirely deleting the thyA gene and replacing it by an inserted functional, wild-type, but promoter-less E. coli lacZ⁺ gene and carrying the 0.8 ribosome binding site (SEQ ID NO: 11) [ΔthyA::(0.8RBS lacZ+)]. In addition to the thyA deletion E866 also carries a deletion of the purA gene. The genotype of strain E866 is given below. E866 incorporates all the changes discussed above and is a host strain suitable for the production of N-acetylglucosamine-containing oligosaccharides.

F′402 proA+B+, PlacIq-/acY, Δ(lacI-lacZ) 158, ΔlacA398/araC, Δgpt-mhpC, ΔthyA::(0.8RBS lacZ+), rpoS+, rph+, ampC::(Ptrp T7g10 RBS-λcl+, CAT), ΔpurA727::KAN

Example 2. Production of N-Acetylglucosamine-Containing Human Milk Oligosaccharides in E. coli: Lacto-N-Tetraose (LNT) and Lacto-N-Neotetraose (LNnT)

The first step in the synthesis (from a lactose precursor) of both Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT) is the addition of a β(1,3)N-acetylglucosamine residue to lactose, utilizing a heterologous β(1,3)-N-acetylglucosaminyltransferase to form Lacto-N-triose 2 (LNT2). The plasmid pG292 (ColE1, thyA+, bla+, P_(L)-lgtA) (SEQ ID NO: 2, FIG. 5) carries the lgtA β(1,3)-N-acetylglucosaminyltransferase gene of N. meningitidis and can direct the production of LNT2 in E. coli strain E680 under appropriate culture conditions. pG221 (ColE1, thyA+, bla+, P_(L)-lgtA-wbgO) (SEQ ID NO: 3, FIG. 6) is a derivative of pG292 that carries (arranged as an operon) both the lgtA β(1,3)-N-acetylglucosaminyltransferase gene of N. meningitidis and the wbgO β(1,3)-galactosyltransferase gene of E. coli O55:H7. pG221 directs the production of LNT in E. coli strain E680 under appropriate culture conditions. pG222 (ColE1, thyA+, bla+, P_(L)-lgtA-4GalT) (SEQ ID NO: 4, FIG. 7) is a derivative of pG292 that carries (arranged as an operon) both the lgtA β(1,3)-N-acetylglucosaminyltransferase gene of N. meningitidis and the 4GalT (jhp0765) β(1,4)-galactosyltransferase gene of H. pylori. pG222 directs the production of LNnT in E. coli strain E680 under appropriate culture conditions.

The addition of tryptophan to the lactose-containing growth medium of cultures of any one of the E680-derivative strains transformed with plasmids pG292, pG221 or pG222 leads, for each particular E680/plasmid combination, to activation of the host E. coli tryptophan utilization repressor TrpR, subsequent repression of P_(trpB), and a consequent decrease in cytoplasmic cI levels, which results in a de-repression of P_(L), expression of lgtA, lgtA+wbgO, or lgtA+4GalT respectively, and production of LNT2, LNT, or LNnT respectively.

For LNT2, LNT, or LNnT production in small scale laboratory cultures (<100 ml), strains were grown at 30° C. in a selective medium lacking both thymidine and tryptophan to early exponential phase (e.g., M9 salts, 0.5% glucose, 0.4% casamino acids). Lactose was then added to a final concentration of 0.5 or 1%, along with tryptophan (200 μM final) to induce expression of the respective glycosyltransferases, driven from the P_(L) promoter. At the end of the induction period (˜24 h), TLC analysis was performed on aliquots of cell-free culture medium. FIG. 8 illustrates schematically the enzymatic reactions necessary to produce from lactose, via the intermediate trisaccharide lacto-N-triose 2 (LNT2), the two human milk oligosaccharides; Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT). A thin layer chromatogram (on left) is presented of culture medium samples taken from small scale E. coli cultures and demonstrating synthesis of LNT2, LNT, and LNnT (utilizing induced, lactose-containing cultures of E680 transformed with pG292, pG221 or pG222 respectively). A second thin layer chromatogram (on right) is presented of culture medium samples taken from an E. coli E680/pG222 15 L bioreactor culture and demonstrating synthesis of LNnT (as well as the higher molecular weight hMOS, Lacto-N-neohexaose, LNnH).

Although the above results clearly demonstrate how it is possible to synthesize GlcNAc-containing oligosaccharides (i.e. LNT2, LNT and LNnT) in engineered E. coli, FIG. 14 illustrates a serious problem faced when attempting to use the E. coli UDP-GlcNAc pool during such syntheses. In FIG. 14 four separate cultures of E680, transformed with pG292, were grown in the presence and absence of lactose, and with LgtA expression both induced and uninduced by tryptophan addition. It can clearly be seen that massive cell lysis occurs in the cultures where lactose is present—i.e. in those cultures where LgtA draws down the cellular UDP-GlcNAc pool by adding GlcNAc to lactose (and making LNT2). In so doing, UDP-GlcNAc is diverted from cell wall biosynthesis towards hMOS biosynthesis, and cell lysis results. This lysis can be monitored readily not only by the precipitous drop in culture density as seen in the figure, but also by the appearance of DNA in the culture medium.

Example 3. Boosting the Cellular UDP-GlcNAc Pool Prevents Cell Lysis During the Biosynthesis of LNnT in Engineered E. coli

To examine the impact of enhancing the E. coli cellular UDP-GlcNAc pool during synthesis of N-acetylglucosamine-containing hMOS the p15A replicon plasmid pG356 was constructed (FIG. 19 and SEQ ID NO:12). pG356 carries a p15A replicon (compatible with ColE1 replicons), purA and ampC selectable markers, and a synthetic operon (under control of the pL promoter) carrying the E. coli glmS (encoding L-glutamine:D-fructose-6-phosphate aminotransferase) and nagC (encoding the bi-functional transcriptional activator/repressor of glm and nag operons) genes. When pL is active in strains carrying the plasmid pG356, the UDP-GlcNAc pool increases. Strain E796 (see example 1) was transformed with pG222 (FIG. 7), and strain E866 (see example 1) was transformed with both pG222 (FIG. 7) and pG356 (FIG. 19). (Strains E796 and E866 are isogenic save for the purA mutation found in E866 that is used for pG356 plasmid retention). Identical 1.5 L fermentation runs were performed on each of the transformed strains. Optical density of the cultures and LNnT biosynthesis was followed, along with standard fermentation parameters. As can be seen in FIG. 18, the E796/pG222 culture produced LNnT, but lysed when the cell density reached 75 OD600, and achieved a final cell density at end-of-fermentation of only 50 OD600. In contrast (FIG. 19) with the E866/pG222+pG356 culture (where expression of the glmS and bagC genes enhance the intracellular UDP-GlcNAc pool) LNnT was also produced, but with no cell lysis observed. In this culture end-of-fermentation cell density reached 108 OD600—more than twice the density achieved for E796/pG222.

Example 4. Production of 6′-Sialyllactose (6′-SL) by Engineered E. coli (ΔnanRATEK)

For the production of 6′ sialyllactose, Escherichia coli GI724 (ATCC55151) was engineered with a set of mutations that cause cytoplasmic accumulation of non-acetylated lactose precursor and prevent the degradation of N-acetyl-5-neuraminic acid (FIG. 3). In particular, the lacZ (β-galactosidase) and lacA (lactose acetyl transferase) genes from the lac operon were deleted, leaving the LacIq repressor and the LacY permease fully functional. The LacY permease can be driven by weak (e.g. lac8) or strong (e.g. Ptac) promoters. The entire nan operon (nanRATEK; structural and regulatory genes involved in neuraminic acid degradation) was deleted in this example. E. coli genome manipulations were achieved using a combination of standard molecular genetics techniques, specifically lambda-Red recombineering, allele exchanges with positive selection suicide vectors, and P1 transductions (FIG. 3). The host genotype of strain E781, suitable for production of sialylated hMOS, is presented below:

ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanRATEK471, ΔlacZ690, ΔlacA 745

To produce 6′-sialyllactose, the cellular UDP-GlcNAc pool must be converted into the sugar-nucleotide activated precursor, CMP-NeuAc, which in turn can function as a donor molecule for a sugar acceptor (i.e. lactose) in a sialyltransferase-catalyzed reaction (FIG. 3). To this purpose, three genes from Campylobacter jejuni ATCC43438, encoding i) UDP-N-acetylglucosamine 2-epimerase (NeuC), ii)N-acetylneuraminate synthase (NeuB), and iii)N-Acetylneuraminate cytidylyltransferase (NeuA), were constitutively co-expressed in the engineered E. coli strain described above, along with a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224 (SEQ ID NO:21 Genbank protein Accession BAF92026, incorporated herein by reference). The neu genes were expressed from a low copy number plasmid vector (pG317, FIG. 9, SEQ ID NO: 5) carrying a constitutive lac promoter (pBBR1 ori, cat+, Plac), while the α(2,6)sialyltransferase gene was expressed from a high copy number plasmid vector (pG315, FIG. 10, SEQ ID NO: 6) carrying a constitutive lac promoter (ColE1 ori, bla+, Plac). To prevent the synthesis of side-products, the relative expression for the α(2,6)sialyltransferase gene compared to the neu genes is modulated by engineering differing ribosomal binding sites (RBS) providing various degrees of translational efficiency upstream of the α(2,6)sialyltransferase gene. Engineered strains were grown to high density in pilot scale fermentors using a batch to fed-batch strategy. FIG. 11 is a TLC analysis of culture supernatants from two such fermentations, with samples to the left of the figure being taken from a fermentation of a strain containing pG315 (and thus carrying the RBS presented in SEQ ID NO: 7 in front of the α(2,6)sialyltransferase gene in the vector). Samples on the right of the figure are taken from a fermentation of a strain containing a close variant of pG315 (pG345, FIG. 12, SEQ ID NO:9, carrying the weaker RBS presented in SEQ ID NO: 8 in front of the α(2,6)sialyltransferase gene and replacing the RBS presented in SEQ ID NO: 7). In both cases, the lactose precursor was added at a cell density of 50 OD₆₀₀ and efficient conversion to final products was achieved within 48 hours from the lactose addition. The final yield of 6′ SL was increased when utilizing the plasmid with the weaker RBS upstream of the α(2,6)sialyltransferase gene, and moreover the level of KDO-lactose side product is very significantly decreased using this weaker RBS. The identity of the 6′-SL purified using activated carbon column chromatography was confirmed by ESI mass spectrometry and NMR.

Example 5. Production of 6′-Sialyllactose (6′-SL) by Engineered E. coli. (ΔnanA, ΔnanATE)

For the production of 6′ sialyllactose, Escherichia coli GI724 (ATCC55151) was engineered with a set of mutations that cause cytoplasmic accumulation of non-acetylated lactose precursor and prevent the degradation of N-acetyl-5-neuraminic acid (FIG. 13). In particular, the lacZ (β-galactosidase) and lacA (lactose acetyl transferase) genes from the lac operon were deleted, leaving the LacIq repressor and the LacY permease fully functional. The LacY permease can be driven by weak (e.g. lac8) or strong (e.g. Ptac) promoters. While the entire nan operon (nanRATEK; structural and regulatory genes involved in neuraminic acid degradation) can be deleted to abolish neuraminic acid catabolism as in Example 4, lesser deletions encompassing just the nanA, or nanA, nanT and nanE, or nanA and nanE genes, are also suitable. In all the instances where the nanE gene was mutated, the last 104 bp of the nanE gene were left intact to allow for undisturbed transcription/translation of downstream nanK, although other lengths of residual nanE sequence are possible. E. coli genome manipulations were achieved using a combination of standard molecular genetics techniques, specifically lambda-Red recombineering, allele exchanges with positive selection suicide vectors, and P1 transductions (FIG. 13). The host genotypes of strains E971, E1017 and E1018, suitable for production of sialylated hMOS with various yield and purity, are presented below:

ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanA::kanR, ΔlacZ690, ΔlacA::scar, ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanATE::kanR::nanK+, ΔlacZ690, ΔlacA::scar and ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanATE::scar::nanK+, ΔlacZ690, ΔlacA::scar respectively

To produce 6′-sialyllactose, the cellular UDP-GlcNAc pool must be converted into the sugar-nucleotide activated precursor, CMP-NeuAc, which in turn can function as a donor molecule for a sugar acceptor (i.e. lactose) in a sialyltransferase-catalyzed reaction (FIG. 13). To this purpose, three genes from Campylobacter jejuni ATCC43438, encoding i) UDP-N-acetylglucosamine 2-epimerase (NeuC), ii)N-acetylneuraminate synthase (NeuB), and iii)N-Acetylneuraminate cytidylyltransferase (NeuA), were constitutively co-expressed in the engineered E. coli strain described above, along with a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224. The neu genes were expressed from a low copy number plasmid vector (pG317, FIG. 9, SEQ ID NO: 5) carrying a constitutive lac promoter (pBBR1 ori, cat+, Plac), while the α(2,6)sialyltransferase gene was expressed from the weak RBS of SEQ ID NO: 8 in a high copy number plasmid vector (pG345, FIG. 12, SEQ ID NO: 9) carrying a constitutive lac promoter (ColE1 ori, bla+, Plac). Engineered strains were grown to high density in pilot scale fermentors using a batch to fed-batch strategy. FIG. 14 is a TLC analysis of culture pellets or supernatants from three such fermentations. Panel A shows production and accumulation of 6′SL in the cells of three genetic backgrounds (only the relevant nan mutations are shown for strains E971, E1017 and E1018), Panel B and C show production and accumulation of 6′SL in the extracellular milieu (supernatants) in strains E971, E1017 and E1018 (only the relevant nan mutations are shown) with estimated maximum volumetric yields of 15 g per liter of supernatant. In all cases, the lactose precursor was added at a cell density of 40 OD₆₀₀ and steady state conversion to final products was achieved within approximately 90 hours from the lactose addition (EFT is elapsed fermentation time).

The various sequences presented herein are recited below.

>E680_thyA::2.8RBS_lacZ Escherichia coli str. SEQ ID NO: 1 GCAGCGGAACTCACAAGGCACCATAACGTCCCCTCCCTGATAACGCTGATACTGTGGTCG CGGTTATGCCAGTTGGCATCTTCACGTAAATAGAGCAAATAGTCCCGCGCCTGGCTGGCG GTTTGCCATAGCCGTTGCGACTGCTGCCAGTATTGCCAGCCATAGAGTCCACTTGCGCTT AGCATGACCAAAATCAGCATCGCGACCAGCGTTTCAATCAGCGTATAACCACGTTGTGTT TTCATGCCGGCAGTATGGAGCGAGGAGAAAAAAAGACGAGGGCCAGTTTCTATTTCTTCG GCGCATCTTCCGGACTATTTACGCCGTTGCAGGACGTTGCAAAATTTCGGGAAGGCGTCT CGAAGAATTTAACGGAGGGTAAAAAAACCGACGCACACTGGCGTCGGCTCTGGCAGGATG TTTCGTAATTAGATAGCCACCGGCGCTTTattaaacctactATGACCATGATTACGGATT CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATC GCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCAC CAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCG TCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATC CCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCA CATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCG TTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTC GTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGG TGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGA GCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCC ATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGA TGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGC AGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATG CCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCC CGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAG AAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACG GCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGG TCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACG CCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACG GCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTC TGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGCAGC GCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCG CTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGT ATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCG TGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGC TACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTC TTGGCGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCT TCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGT CGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTC TGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGT TTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCCGTC ATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAGCG GTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATTGAACTGCCTGAACTAC CGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACGCGA CCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAAAACC TCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCACCAGCGAAATGG ATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTT CACAGATGTGGATTGGCGATAAAAAACAACTGtTGACGCCGCTGCGCGATCAGTTCACCC GTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCT GGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCA CGGCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATCAGG GGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGA TTACCGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGAACT GCCAGCTGGCGCAGGTAGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAGAAAACT ATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGT ATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATT ATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAACAGC AACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATA TCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGG AATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAAGCGG CCGCtTTATGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTT CGGAATAGGAACTTCAAGATCCCCTTATTAGAAGAACTCGTCAAGAAGGCGATAGAAGGC GATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTC GCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGC CACACCCAGCCGGCCACAGTCGATGAATCCtGAAAAGCGGCCATTTTCCACCATGATATT CGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTT GAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTG ATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTG GTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGAT GGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCC CAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAAC GCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACC GGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGC GGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCA AGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCC TGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCAT CCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGG TTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGC TACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATT CATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCA GCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAGCTTCAAAAGCGCTCTGAAGTTCCTAT ACTTTCTAGAGAATAGGAACTTCGAACTGCAGGTCGACGGATCCCCGGAATCATGGTTCC TCAGGAAACGTGTTGCTGTGGGCTGCGACGATATGCCCAGACCATCATGATCACACCCGC GACAATCATCGGGATGGAAAGAATTTGCCCCATGCTGATGTACTGCACCCAGGCACCGGT AAACTGCGCGTCGGGCTGGCGGAAAAACTCAACAATGATGCGAAACGCGCCGTAACCAAT CAGGAACAAACCTGAGACAGCTCCCATTGGGCGTGGTTTACGAATATACAGGTTGAGGAT AATAAACAGCACCACACCTTCCAGCAGCAGCTCGTAAAGCTGTGATGGGTGGCGCGGCAG CACACCGTAAGTGTCGAAAATGGATTGCCACTGCGGGTTGGTTTGCAGCAGCAAAATATC TTCTGTACGGGAGCCAGGGAACAGCATGGCAAACGGGAAGTTCGGGTCAACGCGGCCCCA CAATTCACCGTTAATAAAGTTGCCCAGACGCCCGGCACCAAGACCAAACGGAATGAGTGG TGCGATAAAATCAGAGACCTGGAAGAAGGAACGTTTAGTACGGCGGGCGAAGATAATCAT CACCACGATAACGCCAATCAGGCCGCCGTGGAAAGACATGCCGCCGTCCCAGACACGGAA CAGATACAGCGGATCGGCCATAAACTGCGGGAAATTGTAGAACAGAACATAACCAATACG TCCCCCGAGGAAGACGCCGAGGAAGCCCGCATAGAGTAAGTTTTCAACTTCATTTTTGGT CCAGCCGCTGCCCGGACGATTCGCCCGTCGTGTTGCCAGCCACATTGCAAAAATGAAACC CACCAGATACATCAGGCCGTACCAGTGAAGCGCCACGGGTCCTATTGAGAAAATGACCGG ATCAAACTCCGGAAAATGCAGATAGCTACTGGTCATCTGTCACCACAAGTTCTTGTTATT TCGCTGAAAGAGAACAGCGATTGAAATGCGCGCCGCAGGTTTCAGGCGCTCCAAAGGTGC GAATAATAGCACAAGGGGACCTGGCTGGTTGCCGGATACCGTTAAAAGATATGTATA >pG292, complete sequence. SEQ ID NO: 2 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGCTCGAGTCAACGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGCCTCAA GGTAAACAGCCGCCGCATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCGGGCAG CGTGTCCGTCCGTTTGAAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCTTCTTC CGGCAAATGTTTCTCCAGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCAAGGCT GTCGAACCGGGTTTTAAAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTTTGGAT GCCTTGCGCGATTTCGTGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCGTGAAG GCGGTATTTGACCAAGGCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACATCGTA CCAAAATTGGTAATCTTCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCGTCAAT GACGCTGCGCCTCATAATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAAAAGTC GGCAATGTCTTCGTGTCGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGCCGCGC CAGCCGGTTGCCGTCCTTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCGATGAT GCTGCGGTCTTTTTCCATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCAATATC GTCTGCATCGGTGCGCGCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCATCCAG CCCGATGTTTAAAGAGGGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGGATGCG GCCGTCCTGTTCTTGGAAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCGTCATC GACAATCAAAATATCCAAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGATTGGGC GAAATATTTTTCTACGTTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAATTTATT CTCCCGATAGGCACGATGCCGTCTGAAGGCTTCAGACGGCATATGtatatctccttcttg aaTTCTAACAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAAT TTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTT TTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTT AAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTT CCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGAT TCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCG CGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTAT CACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGA GCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGT ATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTT ATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATT AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGC TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTA GCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA GAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCT CACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTG GTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCA GAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCT GAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAAC TCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG ACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGC CCTTTCGTC >pG221, complete sequence. SEQ ID NO: 3 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGCTCGAGTTATTATTTAATATATTTACAATAGATGAAGGACGCAATCGTACGGAT ACCGCCGAACAGGTAGTTAATGTTACCGGTCAGGAAGAAGCACTTCATTTTGATAACCAG GTCGTTAACCATCACCATGTACAGGTTTTTTTTTGCGGTAGACTGACCTTCGTGCAGGCG GTAGTAGAACAGGTATTCCGGCAGGTTTTGGAACTTGATTTTTGCCAGGCTCAGACGGTT CCACAGCTCGTAATCTTCGGAGTAGTTAGAAAACATATAACCACCGATGCTCGCGATGAC TTTTTTACGAAACATTACGCTCGGGTGAACAATACAACACTTATACGGCAGGTTTTTAAC GATGTCCAGGTTCTCTTCCGGCAGTTTGGTCTTGTTGATTTCACGACCTTTGTCGTCAAT AAAGATTGCGTTGGTACCCACAACATCTACGTACGGATTGTTCTTCAGGAAGTCAACCTG TTTAGTAAAACGGTCCGGGTGAGAGATGTCGTCAGAGTCCATACGGGCAATAAATTCGCC GTTGCTCAGGTCGATCGCTTTGTTCAGGGAGTACGGCAGGTAAGCGATGTTAGTGCGGAT CAGTTTGATTTTGTCGTTAACTTTGTGTTTCAGTTCGTTATAGAAGTCGTCAGTGCAGCA GTTCGCAACGATGATGATTTCGAAGCTGCTGAAGGTCTGAGACAGGATGCTGTTGATCGC TTCGTCCAGAAAAGGGTTTTTCTTGTTAACAGGCAGGATAACGCTCACAACCGGGTGGGT AGATTCCGCGGATTCCGCTTCATCGATGATCATATGTATATCTCCTTCTTCTCGAGTCAA CGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGCCTCAAGGTAAACAGCCGCCGC ATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCGGGCAGCGTGTCCGTCCGTTTG AAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCTTCTTCCGGCAAATGTTTCTCC AGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCAAGGCTGTCGAACCGGGTTTTA AAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTTTGGATGCCTTGCGCGATTTCG TGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCGTGAAGGCGGTATTTGACCAAG GCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACATCGTACCAAAATTGGTAATCT TCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCGTCAATGACGCTGCGCCTCATA ATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAAAAGTCGGCAATGTCTTCGTGT CGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGCCGCGCCAGCCGGTTGCCGTCC TTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCGATGATGCTGCGGTCTTTTTCC ATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCAATATCGTCTGCATCGGTGCGC GCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCATCCAGCCCGATGTTTAAAGAG GGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGGATGCGGCCGTCCTGTTCTTGG AAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCGTCATCGACAATCAAAATATCC AAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGATTGGGCGAAATATTTTTCTACG TTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAATTTATTCTCCCGATAGGCACGA TGCCGTCTGAAGGCTTCAGACGGCATATGtatatctccttcttgaaTTCTAACAATTGAT TGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCA GGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGG GCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGA ACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTC TCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGC TTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGC AGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCC TTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGT GGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCG CCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTT TGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACG CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT ATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTAC CGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGT ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTT ACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG TATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGT AAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAAC TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGG AATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAG CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAA ACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC >pG222, complete sequence. SEQ ID NO: 4 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGctcgagTTATACAAACTGCCAATATTTCAAATATTTAAAATGGAGTTCTCTCAT TAAGGCGATTTTAGGGCTATAAGGTTCTTCTTTTCGTGCTATCGTAGAGATTTGCTCATC ATCAGCGATCACAAAAGGTTGTAACACCAGATTTTTCACGCCATGGATAAAAGTAGCGTC CATTATCGTATCCACAGGAACAACCCATTTTCGGCTGCATTTCAAAAAAACTTTGGCAAT CTTAGGCGTGATCACATAGCCTTGAGTCCCCACCCCTTCGCTATAAGCTTTAATGATCCC CACACGCTCTTGTATCTCGTGGTTTTTATGGCTCAATGGCTCACTTTTTACACTGGCATC ATACAATAAATGCATCAAGCGGATATAGCCTAACTCTTGGATGTGTTTTTCTAAAAAATC CAAGCCCTCTTTAAAATCCTCTTTCAAGGTTATATCGTCTTCTAAAATACAGATCGCTTC ATTGAGTTCTATGCATTTTTCCCACAAGGAATAATGACTCGCATAGCACCCAAGCTCCCC CAAGCTCATAAACTTCGCATGGTATTTTAAAGCGTAATAAAACTTAGAAACCTCACTGAT GAGATTGGTTGTAATCCCCATGTCTTTGATGTTTTGCGTGATGAAATAAGGGTGTAAATG CTTTTTCACTAAGGGGTGCAACCCGCCTTCAAAAGTTTTAGAATAAATCGCATCAAAAAT TTGCGCTTGGTGGTGGGTGGCATTGATGCTATTGAGTAAAGTTGTGGTGTCTCTAAAAAC TAAACCAAATGTATCGCACACTTTTTGATTTAAAGAAATGGCAAAAACACGCAtATGtat atctccttcttCTCGAGTCAACGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGC CTCAAGGTAAACAGCCGCCGCATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCG GGCAGCGTGTCCGTCCGTTTGAAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCT TCTTCCGGCAAATGTTTCTCCAGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCA AGGCTGTCGAACCGGGTTTTAAAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTT TGGATGCCTTGCGCGATTTCGTGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCG TGAAGGCGGTATTTGACCAAGGCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACA TCGTACCAAAATTGGTAATCTTCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCG TCAATGACGCTGCGCCTCATAATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAA AAGTCGGCAATGTCTTCGTGTCGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGC CGCGCCAGCCGGTTGCCGTCCTTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCG ATGATGCTGCGGTCTTTTTCCATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCA ATATCGTCTGCATCGGTGCGCGCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCA TCCAGCCCGATGTTTAAAGAGGGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGG ATGCGGCCGTCCTGTTCTTGGAAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCG TCATCGACAATCAAAATATCCAAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGAT TGGGCGAAATATTTTTCTACGTTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAAT TTATTCTCCCGATAGGCACGATGCCGTCTGAAGGCTTCAGACGGCATATGtatatctcct tcttgaaTTCTAACAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGT TTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTG TTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTA TAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCT GCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATC TGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACC CCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTT CGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTT TAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCA GTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGT TTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCT GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGAT AACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCC ACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAG AAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGT GGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAA TACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG GCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTT CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT TGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC ACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC GAGGCCCTTTCGTC >pG317, complete sequence. SEQ ID NO: 5 GTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCA TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCATGCATAAAAACTGTTGTAATTCA TTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGC GGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAG AAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCT GAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAA CACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTC CAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTA TCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATC AGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTC TTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGAC TGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCA GTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAAT ACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCA ACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGA TTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGAAGACGAAAGGGCCT CGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGG TGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGCGCTGGGCCTGTTTCTGG CGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCCCACGGCCTTGATGATCGCGGCG GCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGC TCCCGAAGGTCTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCT CCTGCGGCGGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCG GCCACGGCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGC GGTGCATAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGC CGCTCCAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATG CCCCGTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATC TGCGCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACC ACGAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGA TCCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGT TCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAAC ACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCG GTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTC TCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTC TTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGGC TCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATACTCA CTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACGGGCTTG CTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATATGTGGA CGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAACCCTGCTG GACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGATTGCCCACCGG CTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCC ATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCT TGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCT TGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAG CCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGGGTTCCAAGGGGGCAGCG CCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAACAAGCCCCGGAGGGGCCACTTT TTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTG GGCACCAAAGAACTAGATATAGGGCGAAATGCGAAAGACTTAAAAATCAACAACTTAAAA AAGGGGGGTACGCAACAGCTCATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAA AGAAAATCTGTAATTGACTGCCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGAA AAGTTGCAGCTGATTGCGCATGGTGCCGCAACCGTGCGGCACCCTACCGCATGGAGATAA GCATGGCCACGCAGTCCAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGG TGCAAACGGAACGCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGG CGGCAATGCTGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCA GCCAGAAGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCA AGGACTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGT CGGCCTACGTGGTCAATGACCGCGTGGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGT CGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGACGACCAGGACGAATCGCTGTTGGGGC ATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCC CCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCCT TGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGATGAGC CGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTGCCGCGCC GGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGC CGCCTCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGG GCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCGTTTTTATCA GGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCT GAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATA AACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGA CCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGC GTAGCACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGC CACTCATCGCAGTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCG AATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTG CTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA CGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTG GCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCG ATACCGTCGACCTCGAGTTAAGTCTCTAATCGATTGTTTTCCAATGGAATGGTTATAAAA TCTTTGGTTTTTAGTCTTGAAAATCTTCTAGGATTTTCTATGTAAGTTTTTGTATAAATA TTATATTGCTTTAATAAATTTAATATATTTTTATTGCATTTTAAGGTTATTTTTTCCATA TCTGTTCAACCTTTTTTAAATCCTCCAAACAGTCAATATCTAAACTTGAGCTTTCGTCCA TTAAAAAATGCTTGGTTTTGCTTTGTAAAAAGCTAGGATTGTTTAAAAATTCTTTTATCT TTAAAATATAAATTGCACCATTGCTCATATAAGTTTTAGGCAATTTTTGCCTTGGCATAA AAGGATATTCATCATTACAAATCCCTGCTAAATCGCCACAATCATTACAAACAAAGGCTT TTAGAATTTTATTATCACATTCGCTTACGCTAATTAGGGCATTTGCATTGCTATTTTTAT AAAGATTAAAAGCTTCATTAATATGAATATTTGTTCTTAGCGGTGAAGTGGGTTGTAAAA AAACTACATCTTCATAATCTTTATAAAATTTTAGAGCATGTAACAGCACTTTATCGCTTG TGGTATCATCTTGTGCAAGGCTAATTGGGCGTTTTAAAATATCAACATTTTGACTTTTTG CATAATTTAAAATTTCATCACTATCACTGCTTACAACAACTTTACTAATGCTTTTAGCAT TTAGTGCAGCTTTGATCGTGTAGTAAATTAAAGGTTTATTGTTTAATAAAACCAAATTTT TATTTTTAATACCCTTTGAGCCACCACGAGCAGGGATTATTGCTAAGCTCATTTTATATC CTTAAAAACTTTTTGTGTGCTGAGTTTAAAAAAATCTCCGCTTTGTAAATATTCAAAAAA TAATTTTGAGCTATCTAAAATCTCTAACTTAGCGCTAAATAAATCTTGTTTTTTATGAAT AGTGTTAATAGCTTTTAGTATTTCATCACTATTTGCATTAACTTTTAGTGTATTTTCATT GCCAAGTCTTCCATTTTGTCTTGAGCCAACTAAAATCCCTGCTGTTTTTAAGTATAAGGC CTCTTTTAAAATACAACTTGAATTACCTATTATAAAATCAGCATTTTTTAACAAAGTTAT AAAATACTCAAATCTAAGCGATGGAAAAAGCTTAAATCTAGGGTTATTTTTAAACTCTTC ATAGCTTTGCAAGATTAATTCAAAACCTAAATCATTATTTGGATAAATAACAATATAATT TTTATTACTTTGTATCAGTGCTTTTACTAAATTGTCTGCTTGATTTTTAATGCTAGTAAT TTCAGTTGTAACAGGATGAAACATAAGCAAAGCGTAGTTTTCATAATTTATATCATAATA TTTTTTTGCTTCGCTAAGTGAAATTTTATTATCGTTTAAAAGTTCTAAATCAGGCGAACC TATGATAAAAATAGATTTTTCATCTTCTCCAAGCTGCATTAAACGCCTTTTTGCAAACTC ATCATTTACTAAATGAATATGAGCTAGTTTTGATATAGCGTGGCGTAAGCTATCGTCAAT AGTTCCTGAAATCTCTCCGCCTTCAATATGCGCTACTAAGATATTATTTAATGCTCCAAC AATAGCTGCTGCTAAAGGCTCAATTCTATCTCCATGTACTACGATTAAATCAGGTTTTAG CTCATTTGCATACCTTGAAAATCCATCAATTGTAGTAGCTAAAGCCTTATCAGTTTGATA ATATTTATCATAATTTATAAATTCATAAATATTTTTAAAGCCATTTTTATAAAGTTCTTT AACTGTATAGCCAAAATTTTTACTTAAGTGCATTCCTGTTGCAAAGATGTAAAGTTCAAA TTCGCTTGAGTTTTGCACCCTGTACATTAAAGATTTAATCTTAGAATAATCAGCCCTAGA GCCTGTTATAAAAAGGATTTTTTTCACGCAAAATCCTCATAGCTTAACTGAGCATCATTT TCTATATCTCTTAATGCTTTTTTGCCTAAAATATTTTCAAATTCAGCCGCACTAATTCCA CCAAGTCCAGGTCTTTTAACCCAAATATTATCCATAGATAAAACTTCGCCTTTTTTAATA TCTTTAATGCTAACTACACTTGCAAAGGCAAAATCAATTGTAACTTGTTCTTGTTTAGCC GCTTTTTTACTTTCATTATTTCCTCTTATTATAGCCATTTGCTCACTTTGTATAATTAGC TCTTTTAAAGCCTTTGTATCCATAGAACAAACTATATCAGGGCCACTTCTATGCATACTA TCAGTAAAATGTCTTTCAAGCACACAAGCTCCAAGTACAACTGCACCTAAACACGCAAGA TTATCTGTTGTGTGGTCGCTTAAGCCTACCATACAAGAAAATTCTTTTTTTAACTCAAGC ATAGCGTTTAATCTTACAAGATTATGCGGGGTTGGGTAAAGATTGGTCGTGTGCATTAAA ACAAAAGGAATTTCATTGTCTAATAAGATTTTTACAGTTGGTTTTATACTTTCAATACTA TTCATTCCTGTGCTAACTATCATAGGCTTTTTAAAGGCTGCTATGTGTTTAATAAGCGGA TAATTATTACACTCACCTGAACCAATCTTAAAAGCACTAACTCCCATATCTTCTAAGCGG TTCGCACCTGCACGAGAAAAAGGTGTGCTAAGATAAACAAGACCTAATTTTTCTGTGTAT TCTTTAAGTGCTAGCTCATCTTTATAATCCAAAGCACATTTTTGCATAATCTCATAAATG CTTATTTTTGCATTACCAGGAATTACTTTTTTAGCGGCCTTACTCATCTCATCTTCAACA ATATGAGTTTGATGCTTTATAATCTTAGCACCTGCGCTAAAGGCTGCATCTACCATAATT TTAGCTAGTTCTAAACTGCCATTATGATTAATGCCTATTTCAGGTACGACTAAGGGTGCT TTTTCTTCACTTATGATTATATTTTGTATTTTTATTTCTTTCATTTATTTTCCTCCTTAG >pG315, complete sequence SEQ ID NO: 6 CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTC ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGA GATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC CTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCG CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCA CCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCTAGACTGCAATACAAACACCTGTTT CACAATTTGGCAGATCAGCCCAAAAAAGTACATTCTCTTCTTTTACAATACCTAGTTTTA TCATTACTTGAACTAAAGGACTTCTCAAAGCAGTTTCACGATCAGTTATAGTTTCTGTCG ATGTAAAAACTATAAATTTAATTTTTTCAGCTGGTATCGTGAAATATAAAGAGCTCGCTA TACCAGCAACTGCATCAGGAAGCATATCTGTCATCATCAAAACTTCAAATGATATTTTTG ATGGAATATCAACCATTGAAGGATAGTTTTGCATTATTAATGTATTAATGATACCGCCAC CAGGGTGACCTTTGAAGAACAAATCATAACTATTGCCTAAATAATGTGGGCTCGATTCAT TAATTGCATTATTAATGACATTAATTTGTTGTTTCGCATAATACTCTCTTTCATGGTTAC CAGCCCATACAGTCGTACCTGTAAACACAAAGTTTGGTAAATTAGATGAATTATATTCAT TTTGTAATTTTTGTTTGTCAAAATTAACAATCGATAAGAATAATTCTTGTTGTTTGCTAT TGAATTTTTTGAAACCATCCCATTGCATTTGCTTTAAACTATCACCAATATAGTCTCGTA ACTCATGTAATGATGGTTCTAAAGTTAAATAATCTTTTCTTAAAAAATGGTAGTTAGCTG GATATAGTTTTTGCCAGTTATAAACAGATGATGTTCCTGTATTTGAAGTGTCTTCATTGA TACCATTAATGACATCCTCAAGATAATCTTTACCAATTTTTAAATTATCTGTTTTATTTA ATGTATCTCTCCAGTTATATAAATTTACATATTCTGCTGAACCATCATCATATAAATCTA TATTTGTTACCGTAACGTTATTAAACGAATTTAATTCTTTTAGTATTGGCACTAAATTAT CAAATGAATGAGCAGTGTTAGAGCTAAGTTTAACATTCAATCTATGCTTTGTTTGTGCTT GCTTAACAATTTCTTGTACTAAGTCAGCTGGTGTATGGTTATTTATCAATGCAAACGATG TAATATTTAACTCTTTCATTTGCTCATCAGTCGGAACTATTCTCCCCCAAGCTATATATC TTTGTGCTGTAGGATTTTCTTCTTCCGATTTAATAATATCCATTAGCTGCTGAAGAGTTG GAAGAGATGCATGATCAACATAAACCTCTAAAGATGGAGCCACTACGTTTAATGTTACTT TTGTTATATATTTTTCACCTTTATTACTAACACCATTAAAATCAAAGCAGTACTTTTCAT CGTCATCTAATCGTGGCGCCACTACAGATAATGATATTGACTCTTTATTTTGTTCTGTTA ATAGTTGTTGCGTACCACAAGTTTGTACCCAAGAGTGTTTTGTAAAAGAGATGTTTGATT GATTAATTGGCTCTAAATTAACATACTCCTCATCAATAATAGTTTTATTAATATCATTTT TAATAATAGATTGTGTATTTTCTTCTGACATggtctgtttcctcCTCGAGGGGGGGCCCG GTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCA TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT GACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC SEQ ID NO: 7 CTCGAGgaggaaacagaccATG SEQ ID NO: 8 CTCGAGgaaagaggggacaaactagATG >pG345, complete sequence SEQ ID NO: 9 CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTC ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGA GATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC CTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCG CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCA CCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCTAGACTGCAATACAAACACCTGTTT CACAATTTGGCAGATCAGCCCAAAAAAGTACATTCTCTTCTTTTACAATACCTAGTTTTA TCATTACTTGAACTAAAGGACTTCTCAAAGCAGTTTCACGATCAGTTATAGTTTCTGTCG ATGTAAAAACTATAAATTTAATTTTTTCAGCTGGTATCGTGAAATATAAAGAGCTCGCTA TACCAGCAACTGCATCAGGAAGCATATCTGTCATCATCAAAACTTCAAATGATATTTTTG ATGGAATATCAACCATTGAAGGATAGTTTTGCATTATTAATGTATTAATGATACCGCCAC CAGGGTGACCTTTGAAGAACAAATCATAACTATTGCCTAAATAATGTGGGCTCGATTCAT TAATTGCATTATTAATGACATTAATTTGTTGTTTCGCATAATACTCTCTTTCATGGTTAC CAGCCCATACAGTCGTACCTGTAAACACAAAGTTTGGTAAATTAGATGAATTATATTCAT TTTGTAATTTTTGTTTGTCAAAATTAACAATCGATAAGAATAATTCTTGTTGTTTGCTAT TGAATTTTTTGAAACCATCCCATTGCATTTGCTTTAAACTATCACCAATATAGTCTCGTA ACTCATGTAATGATGGTTCTAAAGTTAAATAATCTTTTCTTAAAAAATGGTAGTTAGCTG GATATAGTTTTTGCCAGTTATAAACAGATGATGTTCCTGTATTTGAAGTGTCTTCATTGA TACCATTAATGACATCCTCAAGATAATCTTTACCAATTTTTAAATTATCTGTTTTATTTA ATGTATCTCTCCAGTTATATAAATTTACATATTCTGCTGAACCATCATCATATAAATCTA TATTTGTTACCGTAACGTTATTAAACGAATTTAATTCTTTTAGTATTGGCACTAAATTAT CAAATGAATGAGCAGTGTTAGAGCTAAGTTTAACATTCAATCTATGCTTTGTTTGTGCTT GCTTAACAATTTCTTGTACTAAGTCAGCTGGTGTATGGTTATTTATCAATGCAAACGATG TAATATTTAACTCTTTCATTTGCTCATCAGTCGGAACTATTCTCCCCCAAGCTATATATC TTTGTGCTGTAGGATTTTCTTCTTCCGATTTAATAATATCCATTAGCTGCTGAAGAGTTG GAAGAGATGCATGATCAACATAAACCTCTAAAGATGGAGCCACTACGTTTAATGTTACTT TTGTTATATATTTTTCACCTTTATTACTAACACCATTAAAATCAAAGCAGTACTTTTCAT CGTCATCTAATCGTGGCGCCACTACAGATAATGATATTGACTCTTTATTTTGTTCTGTTA ATAGTTGTTGCGTACCACAAGTTTGTACCCAAGAGTGTTTTGTAAAAGAGATGTTTGATT GATTAATTGGCTCTAAATTAACATACTCCTCATCAATAATAGTTTTATTAATATCATTTT TAATAATAGATTGTGTATTTTCTTCTGACATctagtttgtcccctctttcCTCGAGGGGG GGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCA TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATT GCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTC ACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG GTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT AGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTT GGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGG TCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAA AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCG GCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCT GCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGT TCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCA CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC SEQ ID NO: 10 CTTTattaaacctactATG SEQ ID NO: 11 CTTTcttcaacctactATG >pEC3′-(T7)GlmS-(T7)NagC-purA_(pG356) SEQ ID NO: 12 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCG CCactagtGTTGAGGAAAACGATTGGCTGAACAAAAAACAGACTGATCGAGGTCATTTTT GAGTGCAAAAAGTGCTGTAACTCTGAAAAAGCGATGGTAGAATCCATTTTTAAGCAAACG GTGATTTTGAAAAATGGGTAACAACGTCGTCGTACTGGGCACCCAATGGGGTGACGAAGG TAAAGGTAAGATCGTCGATCTTCTGACTGAACGGGCTAAATATGTTGTACGCTACCAGGG CGGTCACAACGCAGGCCATACTCTCGTAATCAACGGTGAAAAAACCGTTCTCCATCTTAT TCCATCAGGTATTCTCCGCGAGAATGTAACCAGCATCATCGGTAACGGTGTTGTGCTGTC TCCGGCCGCGCTGATGAAAGAGATGAAAGAACTGGAAGACCGTGGCATCCCCGTTCGTGA GCGTCTGCTGCTGTCTGAAGCATGTCCGCTGATCCTTGATTATCACGTTGCGCTGGATAA CGCGCGTGAGAAAGCGCGTGGCGCGAAAGCGATCGGCACCACCGGTCGTGGTATCGGGCC TGCTTATGAAGATAAAGTAGCACGTCGCGGTCTGCGTGTTGGCGACCTTTTCGACAAAGA AACCTTCGCTGAAAAACTGAAAGAAGTGATGGAATATCACAACTTCCAGTTGGTTAACTA CTACAAAGCTGAAGCGGTTGATTACCAGAAAGTTCTGGATGATACGATGGCTGTTGCCGA CATCCTGACTTCTATGGTGGTTGACGTTTCTGACCTGCTCGACCAGGCGCGTCAGCGTGG CGATTTCGTCATGTTTGAAGGTGCGCAGGGTACGCTGCTGGATATCGACCACGGTACTTA TCCGTACGTAACTTCTTCCAACACCACTGCTGGTGGCGTGGCGACCGGTTCCGGCCTGGG CCCGCGTTATGTTGATTACGTTCTGGGTATCCTCAAAGCTTACTCCACTCGTGTAGGTGC AGGTCCGTTCCCGACCGAACTGTTTGATGAAACTGGCGAGTTCCTCTGCAAGCAGGGTAA CGAATTCGGCGCAACTACGGGGCGTCGTCGTCGTACCGGCTGGCTGGACACCGTTGCCGT TCGTCGTGCGGTACAGCTGAACTCCCTGTCTGGCTTCTGCCTGACTAAACTGGACGTTCT GGATGGCCTGAAAGAGGTTAAACTCTGCGTGGCTTACCGTATGCCGGATGGTCGCGAAGT GACTACCACTCCGCTGGCAGCTGACGACTGGAAAGGTGTAGAGCCGATTTACGAAACCAT GCCGGGCTGGTCTGAATCCACCTTCGGCGTGAAAGATCGTAGCGGCCTGCCGCAGGCGGC GCTGAACTATATCAAGCGTATTGAAGAGCTGACTGGTGTGCCGATCGATATCATCTCTAC CGGTCCGGATCGTACTGAAACCATGATTCTGCGCGACCCGTTCGACGCGTAATTCTGGTA CGCCTGGCAGATATTTTGCCTGCCGGGCGAACAGTGTGATACATTGCTGTGTCGGGTAAG CCATTACGCTATCCGACACAGTGTTAAATCCTCGCTTTTTTCCTTCCCCagatctGGCGC CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTA TTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAA AAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGT CGACTTAATTTTCCAGCAAATGCTGGAGCAAAATACCGTTGAGCATGGCGCGTTTTACCA GCGCAAAAGCGCCGATTGCCGAGCGGTGATCCAGCTCAGAACGTACCACCGGCAGATTAG TGCGAAACGCCTTCAGCGCCTGGGTATTAATGCAGCTTTCAATAGCAGGGAGCAGCACTT TATCGGCTTCGGTGATTTCACCGGCAATAACAATTTTTTGCGGATTAAATAAGTTGATAG CAATGGCGATGGTTTTACCCAGATGACGACCGACATACTCAATTACTTCCGACGCCAGAC TATCGCCTTTGTTCGCGGCTTTGCAGATAGTTTTGATGGTGCAGTCGTCCAGCGGCACGC GGCTCTGGTAGCCCTGCTTTAACAGATTCAACACCCGTTGTTCAATGGCAGCGTTGGCAG CGATAGTTTCCAGGCAGCCAAAGTTGCCGCAGTGGCAGCGTTCACCCAGCGGTTCGACCT GAATATGGCCAATTTCACCGACGTTGCCGTTGCGGCCAATAAAAATGCGCCCGTTAGAGA TAATCCCGGCCCCGGTTCCGCGATGGACACGCACCAGAATGGAGTCTTCGCAATCCTGAC TTGCACCGAAGTAGTGCTCCGCCAGCGCCAGACTACGGATATCGTGACCAACGAAACAGG TCACTTTAAAACGTTCTTCCAGAGCTTCTACCAGCCCCCAGTTTTCTACCTGAATATGCG GCATGTAATGAATTTTGCCGCTGTCCGGGTCAACAAGCCCTGGCAGGATCACCGAAATCG CGATCAGCTCGCGCAGTTTGCGCTGGTAGCTATCAATAAACTGAGCAATGGCATTCAACA GGGCATGTTCCAGCGTTTGCTGGGTACGTTCCGGCAGCGGGTAATGTTCTTCTGCCAGCA CTTTGCTGCTGAGATCAAACAGAGTGATGGTGGCGTCATGACGACCAAGCCGTACGCCGA TTGCGTGGAAATTGCGGGTTTCGGTGACGATGGAGATAGCGCGGCGGCCCCCGGTGGAGG CCTGCTGATCAACTTCTTTGATCAGCCCGCGTTCGATAAGCTGACGCGTAATTTTGGTTA CGCTGGCGGGGGCAAGCTGGCTTTGCTCGGCAATCTGAATCCGCGAGATTGGCCCGTACT GGTCAATCAGGCGATAAACCGCCGCGCTGTTAAGCTGTTTTACGAGATCAACATTACCTA TCTGAGCTTGTCCGCCTGGTGTCATATGTATATCTCCTTCTTgtcgacTCTAGATGCATG CTCGAGATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCAACGTCGGTGC CTTTGATCAGCGCGACATGGTAAGCCAGCAGCTGCAGCGGAACGGTGTAGAAGATCGGTG CAATCACCTCTTCCACATGCGGCATCTCGATGATGTGCATGTTATCGCTACTTACAAAAC CCGCATCCTGATCGGCGAAGACATACAACTGACCGCCACGCGCGCGAACTTCTTCAATGT TGGATTTCAGTTTTTCCAGCAATTCGTTGTTCGGTGCAACAACAATAACCGGCATATCGG CATCAATTAGCGCCAGCGGACCGTGTTTCAGTTCGCCAGCAGCGTAGGCTTCAGCGTGAA TGTAAGAGATCTCTTTCAACTTCAATGCGCCTTCCAGCGCGATTGGGTACTGATCGCCAC GGCCCAGGAACAGCGCGTGATGTTTGTCAGAGAAATCTTCTGCCAGCGCTTCAATGCGTT TGTCCTGAGACAGCATCTGCTCAATACGGCTCGGCAGCGCCTGCAGACCATGCACGATGT CATGTTCAATGGAGGCATCCAGACCTTTCAGGCGAGACAGCTTCGCCACCAGCATCAACA GCACAGTTAACTGAGTGGTGAATGCTTTAGTGGATGCCACGCCGATTTCTGTACCCGCGT TGGTCATTAGCGCCAGATCGGATTCGCGCACCAGAGAAGAACCCGGAACGTTACAGATTG CCAGTGAACCAAGGTAACCCAGCTCTTTCGACAGACGCAGGCCAGCCAGGGTATCCGCGG TTTCGCCAGACTGTGACAAGGTGATCATCAGGCTGTTACGACGCACGGCAGATTTGCGAT AGCGGAATTCAGAGGCGATTTCGACGTCGCACGGAATACCTGCTAGCGATTCAAACCAGT AGCGGGAAACCATACCGGAGTTATAAGAAGTACCACAGGCGAGGATCTGAATATGCTCAA CCTTCGACAGCAGTTCGTCGGCGTTCGGTCCCAGCTCGCTTAAATCAACCTGACCGTGGC TGATGCGTCCGGTAAGGGTGTTTTTGATCGCGTTCGGCTGTTCGTAGATCTCTTTCTGCA TGTAGTGACGGTAAATGCCTTTATCGCCCGCGTCATATTGCAGATTGGATTCGATATCCT GACGTTTTACTTCCGCGCCAGTTTTATCGAAGATGTTTACCGAACGGCGAGTGATTTCCG CAATATCGCCCTCTTCAAGGAAGATAAAGCGACGGGTCACCGGCAACAGCGCCAGCTGGT CAGAAGCGATAAAGTTTTCGCCCATCCCCAGGCCAATCACCAGCGGACTACCAGAACGTG CCGCCAGCAGGGTATCCGGGTGACGGGAGTCCATGATCACTGTACCGTACGCACCACGCA GCTGCGGGATAGCACGCAGAACGGCCTCACGCAGAGTCCCGCCTTGTTTCAGCTCCCAGT TCACCAGATGGGCAATCACTTCGGTGTCGGTTTCAGAAACGAAGGTATAGCCACGCGCTT TTAGCTCTTCACGCAGCGGTTCATGGTTTTCGATGATGCCGTTATGCACCACCACAATGT GTTCAGAAACATGCGGATGCGCATTCACTTCTGAAGGTTCACCGTGGGTCGCCCAGCGAG TGTGAGCAATACCAGTGCCGCCATGCAGAGGATGTTCTTCCGCTGCCTGTGCCAGCATCT GGACTTTACCGAGGCGACGCAGGCGGGTCATATGACCTTCTGCATCAACAACGGCCAGAC CGGCAGAGTCATATCCGCGGTATTCCAGACGACGTAAACCTTCAAGAAGGATTTCTGCTA CATCACGTTGCGCGATCGCGCCAACAATTCCACACATATGtatatctccttcttgaaTTC TAACAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGAT GCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTT ACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAA AATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATT GAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCC TGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATT GGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACAC ACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTC ACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTA TGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATG AATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTGCTAGCGGA GTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGC AGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCT TCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTAC GAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGG CCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGAC GCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG GCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGT TATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCC AAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGT AATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGAC AAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCA GAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTAC GCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGGC ggccgcGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGG CTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGA TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTT TGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTAT GCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAG AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAA GGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTG AAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAA TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAAC CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC >neuC_N-acetylglucosamine-6-phosphate-2-epimerase_GI_15193223_ in_pG317 SEQ ID NO: 13 MKKILFITGSRADYSKIKSLMYRVQNSSEFELYIFATGMHLSKNFGYTVKELYKNGFKNI YEFINYDKYYQTDKALATTIDGFSRYANELKPDLIVVHGDRIEPLAAAIVGALNNILVAH IEGGEISGTIDDSLRHAISKLAHIHLVNDEFAKRRLMQLGEDEKSIFIIGSPDLELLNDN KISLSEAKKYYDINYENYALLMFHPVTTEITSIKNQADNLVKALIQSNKNYIVIYPNNDL GFELILQSYEEFKNNPRFKLFPSLRFEYFITLLKNADFIIGNSSCILKEALYLKTAGILV GSRQNGRLGNENTLKVNANSDEILKAINTIHKKQDLFSAKLEILDSSKLFFEYLQSGDFF KLSTQKVFKDIK >neuB_sialic_acid_synthase_GI_15193222_in_pG317 SEQ ID NO: 14 MKEIKIQNIIISEEKAPLVVPEIGINHNGSLELAKIMVDAAFSAGAKIIKHQTHIVEDEM SKAAKKVIPGNAKISIYEIMQKCALDYKDELALKEYTEKLGLVYLSTPFSRAGANRLEDM GVSAFKIGSGECNNYPLIKHIAAFKKPMIVSTGMNSIESIKPTVKILLDNEIPFVLMHTT NLYPTPHNLVRLNAMLELKKEFSCMVGLSDHTTDNLACLGAVVLGACVLERHFTDSMHRS GPDIVCSMDTKALKELIIQSEQMAIIRGNNESKKAAKQEQVTIDFAFASVVSIKDIKKGE VLSMDNIWVKRPGLGGISAAEFENILGKKALRDIENDAQLSYEDFA >neuA_CMP-Neu5Ac_synthase_GI_15193224_in_pG317 SEQ ID NO: 15 MSLAIIPARGGSKGIKNKNLVLLNNKPLIYYTIKAALNAKSISKVVVSSDSDEILNYAKS QNVDILKRPISLAQDDTTSDKVLLHALKFYKDYEDVVFLQPTSPLRTNIHINEAFNLYKN SNANALISVSECDNKILKAFVCNDCGDLAGICNDEYPFMPRQKLPKTYMSNGAIYILKIK EFLNNPSFLQSKTKHFLMDESSSLDIDCLEDLKKVEQIWKK >AAF42258 lacto-N-neotetraose biosynthesis glycosyl transferase LgtA [Neisseria meningitidis MC58]. SEQ ID NO: 16 MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTD GTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPD WIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFG NPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQV SSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFER ARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR >NP_207619 lipooligosaccharide 5G8 epitope biosynthesis- associated protein Lex2B [Helicobacter pylori 26695]. SEQ ID NO: 17 MRVFAISLNQKVCDTFGLVFRDTTTLLNSINATHHQAQIFDAIYSKTFEGGLHPLVKKHL HPYFITQNIKDMGITTNLISEVSKFYYALKYHAKFMSLGELGCYASHYSLWEKCIELNEA ICILEDDITLKEDFKEGLDFLEKHIQELGYIRLMHLLYDASVKSEPLSHKNHEIQERVGI IKAYSEGVGTQGYVITPKIAKVFLKCSRKWVVPVDTIMDATFIHGVKNLVLQPFVIADDE QISTIARKEEPYSPKIALMRELHFKYLKYWQFV >E.coli_WbgO_YP_003500090 putative glycosyltransferase WbgO  [Escherichia coli O55:H7 str. CB9615]. SEQ ID NO: 18 MIIDEAESAESTHPVVSVILPVNKKNPFLDEAINSILSQTFSSFEIIIVANCCTDDFYNE LKHKVNDKIKLIRTNIAYLPYSLNKAIDLSNGEFIARMDSDDISHPDRFTKQVDFLKNNP YVDVVGTNAIFIDDKGREINKTKLPEENLDIVKNLPYKCCIVHPSVMFRKKVIASIGGYM FSNYSEDYELWNRLSLAKIKFQNLPEYLFYYRLHEGQSTAKKNLYMVMVNDLVIKMKCFF LTGNINYLFGGIRTIASFIYCKYIK >BAA35319 DNA-binding transcriptional dual regulator nagC  [Escherichia coli str. K-12 substr. W3110]. SEQ ID NO: 19 MTPGGQAQIGNVDLVKQLNSAAVYRLIDQYGPISRIQIAEQSQLAPASVTKITRQLIERG LIKEVDQQASTGGRRAISIVTETRNFHAIGVRLGRHDATITLFDLSSKVLAEEHYPLPER TQQTLEHALLNAIAQFIDSYQRKLRELIAISVILPGLVDPDSGKIHYMPHIQVENWGLVE ALEERFKVTCFVGHDIRSLALAEHYFGASQDCEDSILVRVHRGTGAGIISNGRIFIGRNG NVGEIGHIQVEPLGERCHCGNFGCLETIAANAAIEQRVLNLLKQGYQSRVPLDDCTIKTI CKAANKGDSLASEVIEYVGRHLGKTIAIAINLFNPQKIVIAGEITEADKVLLPAIESCIN TQALKAFRTNLPVVRSELDHRSAIGAFALVKRAMLNGILLQHLLEN >NP_418185 L-glutamtne:D-fructose-6-phosphate aminotransferase glmS [Escherichia coli str. K-12 substr. MG1655]. SEQ ID NO: 20 MCGIVGAIAQRDVAEILLEGLRRLEYRGYDSAGLAVVDAEGHMTRLRRLGKVQMLAQAAE EHPLHGGTGIAHTRWATHGEPSEVNAHPHVSEHIVVVHNGIIENHEPLREELKARGYTFV SETDTEVIAHLVNWELKQGGTLREAVLRAIPQLRGAYGTVIMDSRHPDTLLAARSGSPLV IGLGMGENFIASDQLALLPVTRRFIFLEEGDIAEITRRSVNIFDKTGAEVKRQDIESNLQ YDAGDKGIYRHYMQKEIYEQPNAIKNTLTGRISHGQVDLSELGPNADELLSKVEHIQILA CGTSYNSGMVSRYWFESLAGIPCDVEIASEFRYRKSAVRRNSLMITLSQSGETADTLAGL RLSKELGYLGSLAICNVPGSSLVRESDLALMTNAGTEIGVASTKAFTTQLTVLLMLVAKL SRLKGLDASIEHDIVHGLQALPSRIEQMLSQDKRIEALAEDFSDKHHALFLGRGDQYPIA LEGALKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPNNELLEKLKSNIEEVRAR GGQLYVFADQDAGFVSSDNMHIIEMPHVEEVIAPIFYTVPLQLLAYHVALIKGTDVDQPR NLAKSVTVE >BAF92026 beta-galactoside alpha-2,6-stalyltransferase  [Photobacterium sp. JT-ISH-224]. SEQ ID NO: 21 MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQT CGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVY VDHASLPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADL VQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNL YNWRDTLNKTDNLKIGKDYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTL EPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFT GTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPGGGIINTLIMQNYP SMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRS PLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV Provided below is the DNA sequence in Genbank format of the new configuration of genes engineered at the Escherichia coli thyA locus in strains used to produce N-acetylglucosamine-containing oligosaccharides.

LOCUS E680_thyA::2.8RBS_lacZ 5877 bp DNA linear BCT 04-MAR-2013 DEFINITION Escherichia coli str. K-12 substr. MG1655, complete genome. ACCESSION NC_000913 VERSION NC_000913.2 GI:49175990 KEYWORDS . SOURCE Escherichia coli str. K-12 substr. MG1655 (unknown) ORGANISM Escherichia coli str. K-12 substr. MG1655 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia. REFERENCE 1 (bases 1 to 4639675) AUTHORS Riley, M., Abe, T., Arnaud, M.B., Berlyn, M.K., Blattner, F.R., Chaudhuri, R.R., Glasner, J.D., Horiuchi, T., Keseler, I.M., Kosuge, T., Mori,H., Perna, N.T., Plunkett, G. III, Rudd, K.E., Serres, M.H., Thomas, G.H., Thomson, N.R., Wishart, D. and Wanner, B.L. TITLE Escherichia coli K-12: a cooperatively developed annotation snapshot--2005 JOURNAL Nucleic Acids Res. 34 (1), 1-9 (2006) PUBMED 16397293 REMARK Publication Status: Online-Only REFERENCE 2 (bases 1 to 4639675) AUTHORS Blattner, F.R., Plunkett, G. III, Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B. and Shao, Y. TITLE The complete genome sequence of Escherichia coli K-12 JOURNAL Science 277 (5331), 1453-1474 (1997) PUBMED 9278503 REFERENCE 3 (bases 1 to 4639675) AUTHORS Arnaud, M., Berlyn, M.K.B., Blattner, F.R., Galperin, M.Y., Glasner, J.D., Horiuchi, T., Kosuge, T., Mori, H., Perna, N.T., Plunkett, G. III, Riley, M., Rudd, K.E., Serres, M.H., Thomas, G.H. and Wanner, B.L. TITLE Workshop on Annotation of Escherichia coli K-12 JOURNAL Unpublished REMARK Woods Hole, Mass., on 14-18 November 2003 (sequence corrections) REFERENCE 4 (bases 1 to 4639675) AUTHORS Glasner, J.D., Perna, N.T., Plunkett, G. III, Anderson, B.D., Bockhorst, J., Hu, J.C., Riley, M., Rudd, K.E. and Serres, M.H. TITLE ASAP: Escherichia coli K-12 strain MG1655 version m56 JOURNAL Unpublished REMARK ASAP download 10 June 2004 (annotation updates) REFERENCE 5 (bases 1 to 4639675) AUTHORS Hayashi, K., Morooka, N., Mori, H. and Holiuchi, T. TITLE A more accurate sequence comparison between genomes of Escherichia coli K12 W3110 and MG1655 strains JOURNAL Unpublished REMARK GenBank accessions AG613214 to AG613378 (sequence corrections) REFERENCE 6 (bases 1 to 4639675) AUTHORS Perna, N.T. TITLE Escherichia coli K-12 MG1655 yqiK-rfaE intergenic region, genomic sequence correction JOURNAL Unpublished REMARK GenBank accession AY605712 (sequence corrections) REFERENCE 7 (bases 1 to 4639675) AUTHORS Rudd, K.E. TITLE A manual approach to accurate translation start site annotation: an E. coli K-12 case study JOURNAL Unpublished REFERENCE 8 (bases 1 to 4639675) CONSRTM NCBI Genome Project TITLE Direct Submission JOURNAL Submitted (04-MAR-2013) National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA REFERENCE 9 (bases 1 to 4639675) AUTHORS Rudd, K.E. TITLE Direct Submission JOURNAL Submitted (06-FEB-2013) Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, 118 Gautier Bldg., Miami, FL 33136, USA REMARK Sequence update by submitter REFERENCE 10 (bases 1 to 4639675) AUTHORS Rudd, K.E. TITLE Direct Submission JOURNAL Submitted (24-APR-2007) Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, 118 Gautier Bldg., Miami, FL 33136, USA REMARK Annotation update from ecogene.org as a multi-database collaboration REFERENCE 11 (bases 1 to 4639675) AUTHORS Plunkett, G. III. TITLE Direct Submission JOURNAL Submitted (07-FEB-2006) Laboratory of Genetics, University of Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA REMARK Protein updates by submitter REFERENCE 12 (bases 1 to 4639675) AUTHORS Plunkett, G. III. TITLE Direct Submission JOURNAL Submitted (10-JUN-2004) Laboratory of Genetics, University of Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA REMARK Sequence update by submitter REFERENCE 13 (bases 1 to 4639675) AUTHORS Plunkett, G. III. TITLE Direct Submission JOURNAL Submitted (13-OCT-1998) Laboratory of Genetics, University of Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA REFERENCE 14 (bases 1 to 4639675) AUTHORS Blattner, F.R. and Plunkett, G. III. TITLE Direct Submission JOURNAL Submitted (02-SEP-1997) Laboratory of Genetics, University of Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA REFERENCE 15 (bases 1 to 4639675) AUTHORS Blattner, F.R. and Plunkett, G. III. TITLE Direct Submission JOURNAL Submitted (16-JAN-1997) Laboratory of Genetics, University of Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence is identical to U00096. On Jun 24, 2004 this sequence version replaced gi:16127994. Current U00096 annotation updates are derived from EcoGene ecogene.org. Suggestions for updates can be sent to Dr. Kenneth Rudd (krudd@miami.edu). These updates are being generated from a collaboration that also includes ASAP/ERIC, the Coli Genetic Stock Center, EcoliHub, EcoCyc, RegulonDB and UniProtKB/Swiss- Prot. COMPLETENESS: full length. FEATURES Location/Qualifiers gene complement(<1..245) /gene = ″ppdA″ /locus_tag = ″b2826″ /gene_synonym = ″ECK2822; JW2794″ /db_xref = ″EcoGene:EG12081″ /db_xref = ″GeneID:945393″ CDS complement(<1..245) /gene = ″ppdA″ /locus_tag = ″b2826″ /gene_synonym = ″ECK2822; JW2794″ /function = ″putative enzyme; Not classified″ /GO_component = ″GO:0009289-pilus″ /GO_process = ″GO:0009101-glycoprotein biosynthetic process″ /note = ″prepilin peptidase dependent protein A″ /codon_start = 1 /transl_table = 11 /product = ″hypothetical protein″ /protein_Id = ″NP_417303.1″ /db_xref = ″GI:16130730″ /db_xref = ″ASAP:ABE-0009266″ /db_xref = ″UniProtKB/Swiss-Prot:P33554″ /db_xref = ″EcoGene:EG12081″ /db_xref = ″GeneID:945393″ /translation = ″MKTQRGYTLIETLVAMLILVMLSASGLYGWQYWQQSQRLWQTAS QARDYLLYLREDANWHNRDHSISVIREGTLWCLVSSAAGANTCHGSSPLVFVPRWPEV EMSDLTPSLAFFGLRNTAWAGHIRFKNSTGEWWLVVSPWGRLRLCQQGETEGCL″ (SEQ ID NO: 22) source join(<1..449,4852..>5877) /organism = ″Escherichia coli str. K-12 substr. MG1655″ /mol_type = ″genomic DNA″ /strain = ″K-12″ /sub_strain = ″MG1655″ /db_xref = ″taxon:511145″ primer 346..366 /note = cagtcagtcaggcgccTTCGGGAAGGCGTCTCGAAGA (SEQ ID NO: 23) /label = 0268-THYA-R misc_feature complement(388..394) /feature_type = ″Hairpin loop″ /label = Terminator primer 400..449 /note = GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTag GaaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 24) /label = ″50 bp thyA 3 prime homology″ primer 400..483 /note = GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTat taaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 25) /label = 1389-thyAKANlacZ-R-2-8 primer 400..483 /note = GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTCt tCaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 26) /label = 1516-thyAKANlacZ-R-0-8 primer 400..483 /note = GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTag GaaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 27) /label = ″1041-thyAKANlacZ-R (4-8)″ misc_feature complement(401..407) /feature_type = ″Hairpin loop″ /label = Terminator primer 405..472 /note = CGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTaTTaaac  (SEQ ID NO: 28) ctactATGACCATGAT /label = 1394-2/8-F gene complement(join(429..449,4852..4854)) /gene = ″thyA″ CDS complement(join(429..449,4852..4854)) /gene = ″thyA″ /note = ″ECK2823:JW2795:b2827″ /codon_start = 1 /transl_table = 11 /product = ″thymidylate synthetase″ /protein_Id = ″BAE76896.1″ /db_xref = ″GI:85675643″ /translation = ″MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRFNLQDGFPL VTTKRCHLRSIIHELLWFLQGDTNIAYLHENNVTIWDEWADENGDLGPVYGKQWRAWP TPDGRHIDQITTVLNQLKNDPDSRRIIVSAWNVGELDKMALAPCHAFFQFYVADGKLS CQLYQRSCDVFLGLPFNIASYALLVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQTHL QLSREPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPGIKAPVAI″ (SEQ ID NO: 43) RBS 450..461 /label = ″2.8 RBS″ source 450..3536 /organism = ″Escherichia coli W3110″ /mol_type = ″genomic DNA″ /strain = ″K-12″ /sub_strain = ″W3110″ /db_xref = ″taxon:316407″ /note = ″synonym: Escherichia coli str. K12 substr. W3110″ misc_feature 450..4851 /feature_type = Insertion /note = ″originates from KanR-lacZRBS (E403)″ /label = Insert misc_feature 449{circumflex over ( )}450 /feature_type = ″RBS variation site″ /label = ″C in 0/8″ misc_feature 450..453 /feature_type = ″RBS variation site″ /label = ″CTTC in 0/8″ misc_feature 451..452 /feature_type = ″RBS variation site″ /label = ″GG in 4/8″ misc_feature 451..452 /feature_type = ″RBS variation site″ /label = ″TT in 2/8″ CDS 462..3536 /gene = ″lacZ″ /note = ″ECK0341:JW0335:b0344″ /codon_start = 1 /transl_table = 11 /product = ″beta-D-galactosidase″ /protein_id = ″BAE76126.1″ /db_xref = ″GI:85674486″ /translation = ″MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR TDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYT NVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWV GYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHK PTTQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFG GEIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFR EVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSH YPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRN HPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVD EDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGG FVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQ QQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIE LPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPH LTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSE ATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKT YRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAA CFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETS HRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK″ (SEQ ID NO: 29) primer /label = ″wild-type lacZ+ CDS″ primer complement(1325..1345) /note = TTCAGACGTAGTGTGACGCGA /label = 1042-thyAlacZcheck 2754..2776 /note = TTTCTTTCACAGATGTGGATTGG /label = ″1395-mid lacZ-F″ primer complement(2779..2801) /note = CGGCGTCAGCAGTTGTTTTTTAT /label = ″1396-mid lacZ-R″ mutation 2793 /label = ″C in MG1655 lacZ (silent change)″ Scar complement(3549..3567) /label = ″KD13 downstream scar sequence″ source 3549..4851 /organism = ″Template plasmid pKD13″ /mol_type = ″genomic DNA″ /db_xref = ″taxon:170493″ primer 3549..3568 /label = ″0339 P1w-P2b″ repeat_unit 3568..3579 /label = ″FLP site″ misc_feature complement(3568..3601) /feature_type = ″FRT site″ /label = ″34 bp FRT site″ note complement(3568..4789) /label = ″excised region upon pCP20 introduction″ repeat_unit complement(3590..3601) /label = ″Flp site″ misc_feature complement(3602..3615) /feature_type = ″FRT site″ /note = ″natural FRT site″ /label = ″upstream FRT site″ repeat_unit complement(3604..3615) /label = ″Flp site″ misc_feature complement(3628..4422) /feature_type = ″CDS (KAN resistance)″ /note = ″kanamycin resistance″ /codon_start = 1 /transl_table = 11 /product = ″Tn5 neomycin phosphotransferase″ /protein_id = ″AAL02037.1″ /db_xref = ″GI:15554336″ /translation = ″MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF″ (SEQ ID NO: 30) primer complement(3677..3696) /label = ″0389 KD13_K4″ primer_bind 3791..3810 /label = ″common priming site kt″ primer 3791..3810 /label = ″0344 Wanner Kt primer″ mutation 3811 /label = ″A in wt (silent change)″ primer complement(4242..4261) /label = ″0343 Wanner K2 primer″ primer_bind 4261..4280 /label = ″common priming site k2″ primer_bind 4352..4371 /label = ″common priming site k1″ primer 4352..4371 /label = ″0342 Wanner K1 primer″ repeat_unit 4790..4801 /label = ″FLP site″ Scar complement(4790..4851) /label = ″KD13 upstream scar″ misc_feature complement(4790..4823) /feature_type = ″FRT site″ /label = ″34 bp FRT site″ repeat_unit complement(4812..4823) /label = ″Flp site″ primer complement(4832..4851) /label = ″0338 P4w-P1b″ primer complement(4832..4901) /note = TCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAT TCCGGGGATCCGTCGACC (SEQ ID NO: 31) /label = 1040-thyAKANlacZ-F Site complement(4858..4863) /site_type = ″binding site″ /label = ″thyA RBS″ gene complement(4861..5736) /gene = ″lgt″ CDS complement(4861..5736) /gene = ″lgt″ /note = ″ECK2824:JW2796:b2828″ /codon_start = 1 /transl_tab1e = 11 /product = ″phosphatidylglycerol-prolipoprotein diacylglyceryl transferase″ /protein_Id = ″BAE76897.1″ /db_xref = ″GI:85675644″ /translation = ″MTSSYLHFPEFDPVIFSIGPVALHWYGLMYLVGFIFAMWLATRR ANRPGSGWTKNEVENLLYAGFLGVFLGGRIGYVLFYNFPQFMADPLYLFRVWDGGMSF HGGLIGVIVVMIIFARRTKRSFFQVSDFIAPLIPFGLGAGRLGNFINGELWGRVDPNF PFAMLFPGSRTEDILLLQTNPQWQSIFDTYGVLPRHPSQLYELLLEGVVLFIILNLYI RKPRPMGAVSGLFLIGYGAFRIIVEFFRQPDAQFTGAWVQYISMGQILSIPMIVAGVI MMVWAYRRSPQQHVS″ (SEQ ID NO: 32) promoter complement(4957..4962) /label = ″thyA WEAK -10″ promoter complement(4978..4983) /label = ″thyA -35″ primer complement(5076..5099) /note = cagtcagtcaggcgccTCCTCAACCTGTATATTCGTAAAC (SEQ ID NO: 33) /label = 0267-THYA-F Site complement(5739..5744) /site_type = ″binding site″ /label = ″Igt RBS″ promoter complement(5823..5828) /label = ″Igt -10 (strong)″ ORIGIN    1 GCAGCGGAAC TCACAAGGCA CCATAACGTC CCCTCCCTGA TAACGCTGAT ACTGTGGTCG   61 CGGTTATGCC AGTTGGCATC TTCACGTAAA TAGAGCAAAT AGTCCCGCGC CTGGCTGGCG  121 GTTTGCCATA GCCGTTGCGA CTGCTGCCAG TATTGCCAGC CATAGAGTCC ACTTGCGCTT  181 AGCATGACCA AAATCAGCAT CGCGACCAGC GTTTCAATCA GCGTATAACC ACGTTGTGTT  241 TTCATGCCGG CAGTATGGAG CGAGGAGAAA AAAAGACGAG GGCCAGTTTC TATTTCTTCG  301 GCGCATCTTC CGGACTATTT ACGCCGTTGC AGGACGTTGC AAAATTTCGG GAAGGCGTCT  361 CGAAGAATTT AACGGAGGGT AAAAAAACCG ACGCACACTG GCGTCGGCTC TGGCAGGATG  421 TTTCGTAATT AGATAGCCAC CGGCGCTTTa ttaaacctac tATGACCATG ATTACGGATT  481 CACTGGCCGT CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC CAACTTAATC  541 GCCTTGCAGC ACATCCCCCT TTCGCCAGCT GGCGTAATAG CGAAGAGGCC CGCACCGATC  601 GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGCG CTTTGCCTGG TTTCCGGCAC  661 CAGAAGCGGT GCCGGAAAGC TGGCTGGAGT GCGATCTTCC TGAGGCCGAT ACTGTCGTCG  721 TCCCCTCAAA CTGGCAGATG CACGGTTACG ATGCGCCCAT CTACACCAAC GTGACCTATC  781 CCATTACGGT CAATCCGCCG TTTGTTCCCA CGGAGAATCC GACGGGTTGT TACTCGCTCA  841 CATTTAATGT TGATGAAAGC TGGCTACAGG AAGGCCAGAC GCGAATTATT TTTGATGGCG  901 TTAACTCGGC GTTTCATCTG TGGTGCAACG GGCGCTGGGT CGGTTACGGC CAGGACAGTC  961 GTTTGCCGTC TGAATTTGAC CTGAGCGCAT TTTTACGCGC CGGAGAAAAC CGCCTCGCGG 1021 TGATGGTGCT GCGCTGGAGT GACGGCAGTT ATCTGGAAGA TCAGGATATG TGGCGGATGA 1081 GCGGCATTTT CCGTGACGTC TCGTTGCTGC ATAAACCGAC TACACAAATC AGCGATTTCC 1141 ATGTTGCCAC TCGCTTTAAT GATGATTTCA GCCGCGCTGT ACTGGAGGCT GAAGTTCAGA 1201 TGTGCGGCGA GTTGCGTGAC TACCTACGGG TAACAGTTTC TTTATGGCAG GGTGAAACGC 1261 AGGTCGCCAG CGGCACCGCG CCTTTCGGCG GTGAAATTAT CGATGAGCGT GGTGGTTATG 1321 CCGATCGCGT CACACTACGT CTGAACGTCG AAAACCCGAA ACTGTGGAGC GCCGAAATCC 1381 CGAATCTCTA TCGTGCGGTG GTTGAACTGC ACACCGCCGA CGGCACGCTG ATTGAAGCAG 1441 AAGCCTGCGA TGTCGGTTTC CGCGAGGTGC GGATTGAAAA TGGTCTGCTG CTGCTGAACG 1501 GCAAGCCGTT GCTGATTCGA GGCGTTAACC GTCACGAGCA TCATCCTCTG CATGGTCAGG 1561 TCATGGATGA GCAGACGATG GTGCAGGATA TCCTGCTGAT GAAGCAGAAC AACTTTAACG 1621 CCGTGCGCTG TTCGCATTAT CCGAACCATC CGCTGTGGTA CACGCTGTGC GACCGCTACG 1681 GCCTGTATGT GGTGGATGAA GCCAATATTG AAACCCACGG CATGGTGCCA ATGAATCGTC 1741 TGACCGATGA TCCGCGCTGG CTACCGGCGA TGAGCGAACG CGTAACGCGA ATGGTGCAGC 1801 GCGATCGTAA TCACCCGAGT GTGATCATCT GGTCGCTGGG GAATGAATCA GGCCACGGCG 1861 CTAATCACGA CGCGCTGTAT CGCTGGATCA AATCTGTCGA TCCTTCCCGC CCGGTGCAGT 1921 ATGAAGGCGG CGGAGCCGAC ACCACGGCCA CCGATATTAT TTGCCCGATG TACGCGCGCG 1981 TGGATGAAGA CCAGCCCTTC CCGGCTGTGC CGAAATGGTC CATCAAAAAA TGGCTTTCGC 2041 TACCTGGAGA GACGCGCCCG CTGATCCTTT GCGAATACGC CCACGCGATG GGTAACAGTC 2101 TTGGCGGTTT CGCTAAATAC TGGCAGGCGT TTCGTCAGTA TCCCCGTTTA CAGGGCGGCT 2161 TCGTCTGGGA CTGGGTGGAT CAGTCGCTGA TTAAATATGA TGAAAACGGC AACCCGTGGT 2221 CGGCTTACGG CGGTGATTTT GGCGATACGC CGAACGATCG CCAGTTCTGT ATGAACGGTC 2281 TGGTCTTTGC CGACCGCACG CCGCATCCAG CGCTGACGGA AGCAAAACAC CAGCAGCAGT 2341 TTTTCCAGTT CCGTTTATCC GGGCAAACCA TCGAAGTGAC CAGCGAATAC CTGTTCCGTC 2401 ATAGCGATAA CGAGCTCCTG CACTGGATGG TGGCGCTGGA TGGTAAGCCG CTGGCAAGCG 2461 GTGAAGTGCC TCTGGATGTC GCTCCACAAG GTAAACAGTT GATTGAACTG CCTGAACTAC 2521 CGCAGCCGGA GAGCGCCGGG CAACTCTGGC TCACAGTACG CGTAGTGCAA CCGAACGCGA 2581 CCGCATGGTC AGAAGCCGGG CACATCAGCG CCTGGCAGCA GTGGCGTCTG GCGGAAAACC 2641 TCAGTGTGAC GCTCCCCGCC GCGTCCCACG CCATCCCGCA TCTGACCACC AGCGAAATGG 2701 ATTTTTGCAT CGAGCTGGGT AATAAGCGTT GGCAATTTAA CCGCCAGTCA GGCTTTCTTT 2761 CACAGATGTG GATTGGCGAT AAAAAACAAC TGtTGACGCC GCTGCGCGAT CAGTTCACCC 2821 GTGCACCGCT GGATAACGAC ATTGGCGTAA GTGAAGCGAC CCGCATTGAC CCTAACGCCT 2881 GGGTCGAACG CTGGAAGGCG GCGGGCCATT ACCAGGCCGA AGCAGCGTTG TTGCAGTGCA 2941 CGGCAGATAC ACTTGCTGAT GCGGTGCTGA TTACGACCGC TCACGCGTGG CAGCATCAGG 3001 GGAAAACCTT ATTTATCAGC CGGAAAACCT ACCGGATTGA TGGTAGTGGT CAAATGGCGA 3061 TTACCGTTGA TGTTGAAGTG GCGAGCGATA CACCGCATCC GGCGCGGATT GGCCTGAACT 3121 GCCAGCTGGC GCAGGTAGCA GAGCGGGTAA ACTGGCTCGG ATTAGGGCCG CAAGAAAACT 3181 ATCCCGACCG CCTTACTGCC GCCTGTTTTG ACCGCTGGGA TCTGCCATTG TCAGACATGT 3241 ATACCCCGTA CGTCTTCCCG AGCGAAAACG GTCTGCGCTG CGGGACGCGC GAATTGAATT 3301 ATGGCCCACA CCAGTGGCGC GGCGACTTCC AGTTCAACAT CAGCCGCTAC AGTCAACAGC 3361 AACTGATGGA AACCAGCCAT CGCCATCTGC TGCACGCGGA AGAAGGCACA TGGCTGAATA 3421 TCGACGGTTT CCATATGGGG ATTGGTGGCG ACGACTCCTG GAGCCCGTCA GTATCGGCGG 3481 AATTCCAGCT GAGCGCCGGT CGCTACCATT ACCAGTTGGT CTGGTGTCAA AAATAAGCGG 3541 CCGCtTTATG TAGGCTGGAG CTGCTTCGAA GTTCCTATAC TTTCTAGAGA ATAGGAACTT 3601 CGGAATAGGA ACTTCAAGAT CCCCTTATTA GAAGAACTCG TCAAGAAGGC GATAGAAGGC 3661 GATGCGCTGC GAATCGGGAG CGGCGATACC GTAAAGCACG AGGAAGCGGT CAGCCCATTC 3721 GCCGCCAAGC TCTTCAGCAA TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC 3781 CACACCCAGC CGGCCACAGT CGATGAATCC tGAAAAGCGG CCATTTTCCA CCATGATATT 3841 CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA TGCGCGCCTT 3901 GAGCCTGGCG AACAGTTCGG CTGGCGCGAG CCCCTGATGC TCTTCGTCCA GATCATCCTG 3961 ATCGACAAGA CCGGCTTCCA TCCGAGTACG TGCTCGCTCG ATGCGATGTT TCGCTTGGTG 4021 GTCGAATGGG CAGGTAGCCG GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT 4081 GGATACTTTC TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC 4141 CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG CGCAAGGAAC 4201 GCCCGTCGTG GCCAGCCACG ATAGCCGCGC TGCCTCGTCC TGCAGTTCAT TCAGGGCACC 4261 GGACAGGTCG GTCTTGACAA AAAGAACCGG GCGCCCCTGC GCTGACAGCC GGAACACGGC 4321 GGCATCAGAG CAGCCGATTG TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA 4381 AGCGGCCGGA GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC 4441 TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA AGAAAGCCAT 4501 CCAGTTTACT TTGCAGGGCT TCCCAACCTT ACCAGAGGGC GCCCCAGCTG GCAATTCCGG 4561 TTCGCTTGCT GTCCATAAAA CCGCCCAGTC TAGCTATCGC CATGTAAGCC CACTGCAAGC 4621 TACCTGCTTT CTCTTTGCGC TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT 4681 CATCCGGGGT CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA 4741 GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAGCTTCAA AAGCGCTCTG AAGTTCCTAT 4801 ACTTTCTAGA GAATAGGAAC TTCGAACTGC AGGTCGACGG ATCCCCGGAA TCATGGTTCC 4861 TCAGGAAACG TGTTGCTGTG GGCTGCGACG ATATGCCCAG ACCATCATGA TCACACCCGC 4921 GACAATCATC GGGATGGAAA GAATTTGCCC CATGCTGATG TACTGCACCC AGGCACCGGT 4981 AAACTGCGCG TCGGGCTGGC GGAAAAACTC AACAATGATG CGAAACGCGC CGTAACCAAT 5041 CAGGAACAAA CCTGAGACAG CTCCCATTGG GCGTGGTTTA CGAATATACA GGTTGAGGAT 5101 AATAAACAGC ACCACACCTT CCAGCAGCAG CTCGTAAAGC TGTGATGGGT GGCGCGGCAG 5161 CACACCGTAA GTGTCGAAAA TGGATTGCCA CTGCGGGTTG GTTTGCAGCA GCAAAATATC 5221 TTCTGTACGG GAGCCAGGGA ACAGCATGGC AAACGGGAAG TTCGGGTCAA CGCGGCCCCA 5281 CAATTCACCG TTAATAAAGT TGCCCAGACG CCCGGCACCA AGACCAAACG GAATGAGTGG 5341 TGCGATAAAA TCAGAGACCT GGAAGAAGGA ACGTTTAGTA CGGCGGGCGA AGATAATCAT 5401 CACCACGATA ACGCCAATCA GGCCGCCGTG GAAAGACATG CCGCCGTCCC AGACACGGAA 5461 CAGATACAGC GGATCGGCCA TAAACTGCGG GAAATTGTAG AACAGAACAT AACCAATACG 5521 TCCCCCGAGG AAGACGCCGA GGAAGCCCGC ATAGAGTAAG TTTTCAACTT CATTTTTGGT 5581 CCAGCCGCTG CCCGGACGAT TCGCCCGTCG TGTTGCCAGC CACATTGCAA AAATGAAACC 5641 CACCAGATAC ATCAGGCCGT ACCAGTGAAG CGCCACGGGT CCTATTGAGA AAATGACCGG 5701 ATCAAACTCC GGAAAATGCA GATAGCTACT GGTCATCTGT CACCACAAGT TCTTGTTATT 5761 TCGCTGAAAG AGAACAGCGA TTGAAATGCG CGCCGCAGGT TTCAGGCGCT CCAAAGGTGC 5821 GAATAATAGC ACAAGGGGAC CTGGCTGGTT GCCGGATACC GTTAAAAGAT ATGTATA (SEQ ID NO: 34) // Provided below is the DNA sequence in Genbank format of the configuration of genes at the Escherichia coli nan locus, and the details of the deletion endpoints found in engineered strains E1017 and E1018.

LOCUS W3110_nanRATEKyhcH_region 5861 bp DNA linear BCT 19-FEB-2009 DEFINITION Escherichia coli str. K-12 substr. W3110 strain K-12. ACCESSION AC_000091 VERSION AC_000091.1 GI:89106884 KEYWORDS . SOURCE Escherichia coli str. K-12 substr. W3110 (unknown) ORGANISM Escherichia coli str. K-12 substr. W3110 Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia. REFERENCE 1 AUTHORS Riley, M., Abe, T., Arnaud, M.B., Berlyn, M.K., Blattner, F.R., Chaudhuri, R.R., Glasner, J.D., Horiuchi, T., Keseler, I.M., Kosuge, T., Mori, H., Perna, N.T., Plunkett, G. III, Rudd, K.E., Serres, M.H., Thomas, G.H., Thomson, N.R., Wishart, D. and Wanner, B.L. TITLE Escherichia coli K-12: a cooperatively developed annotation snapshot--2005 JOURNAL Nucleic Acids Res. 34 (1), 1-9 (2006) PUBMED 16397293 REMARK Publication Status: Online-Only REFERENCE 2 (bases 1 to 4646332) AUTHORS Hayashi, K., Morooka, N., Yamamoto, Y., Fujita, K., Isono, K., Choi, S., Ohtsubo, E., Baba, T., Wanner, B.L., Mori, H. and Horiuchi, T. TITLE Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110 JOURNAL Mol. Syst. Bio. 2, 2006 (2006) PUBMED 16738553 REFERENCE 3 AUTHORS Yamamoto, Y., Aiba, H., Baba, T., Hayashi, K., Inada, T., Isono, K., Itoh, T., Kimura, S., Kitagawa, M., Makino, K., Miki, T., Mitsuhashi, N., Mizobuchi, K., Mori, H., Nakade, S., Nakamura, Y., Nashimoto,H., Oshima, T., Oyama, S., Saito, N., Sampei, G., Satoh, Y., Siyasundaram, S., Tagami, H., Takahashi, H., Takeda,J., Takemoto, K., Uehara, K., Wada, C., Yamagata, S. and Horiuchi, T. TITLE Construction of a contiguous 874-kb sequence of the Escherichia coli-K12 genome corresponding to 50.0-68.8 min on the linkage map and analysis of its sequence features JOURNAL DNA Res. 4 (2), 91-113 (1997) PUBMED 9205837 REFERENCE 4 AUTHORS Itoh, T., Aiba, H., Baba, T., Hayashi, K., Inada, T., Isono, K., Kasai, H., Kimura, S., Kitakawa, M., Kitagawa, M., Makino, K., Miki, T., Mizobuchi, K., Mori, H., Mori, T., Motomura, K., Nakade, S., Nakamura, Y., Nashimoto, H., Nishio, Y., Oshima, T., Saito, N., Sampei, G., Seki, Y., Siyasundaram, S., Tagami, H., Takeda, J., Takemoto, K., Wada, C., Yamamoto, Y. and Horiuchi, T. TITLE A 460-kb DNA sequence of the Escherichia coil K-12 genome corresponding to the 40.1-50.0 min region on the linkage map JOURNAL DNA Res. 3 (6), 379-392 (1996) PUBMED 9097040 REFERENCE 5 AUTHORS Aiba, H., Baba, T., Hayashi, K., Inada, T., Isono, K., Itoh, T., Kasai, H., Kashimoto, K., Kimura, S., Kitakawa, M., Kitagawa, M., Makino, K., Miki, T., Mizobuchi, K., Mori, H., Mori,T., Motomura, K., Nakade, S., Nakamura, Y., Nashimoto, H., Nishio, Y., Oshima, T., Saito, N., Sampei, G., Seki, Y., Siyasundaram, S., Tagami, H., Takeda, J., Takemoto, K., Takeuchi, Y., Wada, C., Yamamoto, Y. and Horiuchi, T. TITLE A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map JOURNAL DNA Res. 3 (6), 363-377 (1996) PUBMED 9097039 REFERENCE 6 AUTHORS Arn, E.A. and Abelson, J.N. TITLE The 2′-5′ RNA ligase of Escherichia coli Purification, cloning, and genomic disruption JOURNAL J. Bio. Chem. 271 (49), 31145-31153 (1996) PUBMED 8940112 REFERENCE 7 AUTHORS Oshima, T., Aiba, H., Baba, T., Fujita, K., Hayashi, K., Honjo, A., Ikemoto, K., Inada, T., Itoh, T., Kajihara, M., Kanai, K., Kashimoto, K., Kimura, S., Kitagawa, M., Makino, K., Masuda, S., Miki, T., Mizobuchi, K., Mori, H., Motomura, K., Nakamura, Y., Nashimoto, H., Nishio, Y., Saito, N., Sampei, G., Seki, Y., Tagami, H., Takemoto, K., Wada, C., Yamamoto, Y., Yano, M. and Horlichi, T. TITLE A 718-kb DNA  sequence of the Escherichia coli K-12 genome corresponding to the 12.7-28.0 min region on the linkage map JOURNAL DNA Res. 3 (3), 137-155 (1996) PUBMED 8905232 REFERENCE 8 AUTHORS Fujita, N., Mori,H., Yura, T. and Ishliama, A. TITLE Systematic sequencing of the Escherichia coli genome: analysis of the 2.4-4.1 min (110,917-193,643 bp) region JOURNAL Nucleic Acids Res. 22 (9), 1637-1639 (1994) PUBMED 8202364 REFERENCE 9 AUTHORS Janosi, L., Shimizu, I. and Kaji, A. TITLE Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth JOURNAL Proc. Natl. Acad. Sci. U.S.A. 91 (10), 4249-4253 (1994) PUBMED 8183897 REFERENCE 10 AUTHORS Allikmets, R., Gerrard, B., Court, D. and Dean, M. TITLE Cloning and organization of the abc and mdl genes of Escherichia coli: relationship to eukaryotic multidrug resistance JOURNAL Gene 136 (1-2), 231-236 (1993) PUBMED 7904973 REFERENCE 11 AUTHORS van Heeswijk, W.C., Rabenberg, M., Westerhoff, H.V. and Kahn, D. TITLE The genes of the glutamine synthetase adenylylation cascade; 13 are not regulated by nitrogen in Escherichia coli JOURNAL Mol. Microbiol. 9 (3), 443-457 (1993) PUBMED 8412694 REFERENCE 12 AUTHORS Zhao, S., Sandt, C.H., Feulner, G., Vlazny, D.A., Gray, J.A. and Hill, C.W. TITLE Rhs elements of Escherichia coli K-12: complex composites of shared and unique components that have different evolutionary 14) histories JOURNAL J. Bacteriol. 175 (10), 2799-2808 (1993) PUBMED 8387990 REFERENCE 13 AUTHORS Yamada, M., Asaoka, S., Saier, M.H. Jr. and Yamada, Y. TITLE Characterization of the gcd gene from Escherichia coli K-12 W3110 and regulation of its expression JOURNAL J. Bacteriol. 175 (2), 568-571 (1993) PUBMED 8419307 REFERENCE 14 AUTHORS Cormack, R.S. and Mackie, G.A. TITLE Structural requirements for the processing of Escherichia coli 5 S ribosomal RNA by RNase E in vitro JOURNAL J. Mol. Bio. 228 (4), 1078-1090 (1992) PUBMED 1474579 REFERENCE 15 AUTHORS Gervali, F.G. and Drapeau, G.R. TITLE Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12 JOURNAL J. Bacteriol. 174 (24), 8016-8022 (1992) PUBMED 1459951 REFERENCE 16 AUTHORS Yamanaka, K., Ogura, T., Niki, H. and Hiraga, S. TITLE Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli JOURNAL J. Bacteriol. 174 (23), 7517-7526 (1992) PUBMED 1447125 REFERENCE 17 AUTHORS Condon, C., Philips, J., Fu, Z.Y., Squires, C. and Squires, C.L. TITLE Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli JOURNAL EMBO J. 11 (11), 4175-4185 (1992) PUBMED 1396599 REFERENCE 18 AUTHORS Arnqvist, A., Olsen, A., Pfeifer, J., Russell, D.G. and Normark, S. TITLE The Crl protein activates cryptic genes for curl formation and fibronectin binding in Esherichia coli HB101 JOURNAL Mol. Microbiol. 6 (17), 2443-2452 (1992) PUBMED 1357528 REFERENCE 19 AUTHORS Talarico, T.L., Ray, P.H., Dev, I.K., Merrill, B.M. and Dallas, W.S. TITLE Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin- pyrophosphokinase JOURNAL J. Bacteriol. 174 (18), 5971-5977 (1992) PUBMED 1325970 REFERENCE 20 AUTHORS LI, S.J. and Cronan, J.E. Jr. TITLE The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase JOURNAL J. Bio. Chem. 267 (24), 16841-16847 (1992) PUBMED 1355089 REFERENCE 21 AUTHORS Yura, T., Mori, H., Nagai, H., Nagata, T., Ishihama, A., Fujita,N., Isono, K., Mizobuchi, K. and Nakata, A. TITLE Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region JOURNAL Nucleic Acids Res. 20 (13), 3305-3308 (1992) PUBMED 1630901 REFERENCE 22 AUTHORS Ghosh, S.K., Biswas, S.K., Paul, K. and Das, J. TITLE Nucleotide and deduced amino acid sequence of the recA gene of Vibrio cholerae JOURNAL Nucleic Acids Res. 20 (2), 372 (1992) PUBMED 1741267 REFERENCE 23 AUTHORS Smallshaw, J.E. and Kelln, R.A. TITLE Cloning, nucleotide sequence and expression of the Escherichia coli K-12 pyrH gene encoding UMP kinase JOURNAL Genetics (Life Sci. Adv.) 11, 59-65 (1992) REFERENCE 24 AUTHORS O'Neill, G.P., Grygorczyk, R., Adam, M. and Ford-Hutchinson, A.W. TITLE The nucleotide sequence of a voltage-gated chloride channel from the electric organ of Torpedo californica JOURNAL Biochim. Biophys. Acta 1129 (1), 131-134 (1991) PUBMED 1721838 REFERENCE 25 AUTHORS Kajie, S., Ideta, R., Yamato, I. and Anraku, Y. TITLE Molecular cloning and DNA sequence of dniR, a gene affecting anaerobic expression of the Escherichia coli hexaheme nitrite reductase JOURNAL FEMS Microbiol. Lett. 67 (2), 205-211 (1991) PUBMED 1663890 REFERENCE 26 AUTHORS Hershfield, M.S., Chaffee, S., Koro-Johnson, L., Mary, A., Smith, A.A. and Short, S.A. TITLE Use of site-directed mutagenesis to enhance the epitope- shielding effect of covalent modification of proteins with polyethylene glycol JOURNAL Proc. Natl. Acad. Sci. U.S.A. 88 (16), 7185-7189 (1991) PUBMED 1714590 REFERENCE 27 AUTHORS Shimizu, I. and Kaji, A. TITLE Identification of the promoter region of the ribosome- releasing factor cistron (frr) JOURNAL J. Bacteriol. 173 (16), 5181-5187 (1991) PUBMED 1860827 REFERENCE 28 AUTHORS Poulsen, L.K., Refn, A., Molin, S. and Andersson, P. TITLE The gef gene from Escherichia coli is regulated at the level of translation JOURNAL Mol. Microbiol. 5 (7), 1639-1648 (1991) PUBMED 1943701 REFERENCE 29 AUTHORS Poulsen, L.K., Refn, A., Molin, S. and Andersson, P. TITLE Topographic analysis of the toxic Gef protein from Escherichia coli  JOURNAL Mol. Microbiol. 5 (7), 1627-1637 (1991) PUBMED 1943700 REFERENCE 30 AUTHORS Kawamukai, M., Utsumi, R., Takeda, K., Higashi, A., Matsuda, H., Choi, Y.L. and Komano, T. TITLE Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved In CRP*-dependent mal gene expression in Escherichia coli JOURNAL J. Bacteriol. 173 (8), 2644-2648 (1991) PUBMED 2013578 REFERENCE 31 AUTHORS Hulton, C.S., Higgins, C.F. and Sharp, P.M. TITLE ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli Salmonella typhimurium and other enterobacteria JOURNAL Mol. Microbiol. 5 (4), 825-834 (1991) PUBMED 1713281 REFERENCE 32 AUTHORS Munro, A.W., Ritchie, G.Y., Lamb, A.J., Douglas, R.M. and Booth, I.R. TITLE The cloning and DNA sequence of the gene for the glutathione-regulated potassium-efflux system KefC of Escherichia coli JOURNAL Mol. Microbiol. 5 (3), 607-616 (1991) PUBMED 2046548 REFERENCE 33 AUTHORS Arigoni, F., Kaminski, P.A., Hennecke, H. and Elmerich, C. TITLE Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW JOURNAL Mol. Gen. Genet. 225 (3), 514-520 (1991) PUBMED 1850088 REFERENCE 34 AUTHORS Mattick, J.S., Anderson, B.J., Cox, P.T., Dalrymple, B.P., Bills, M.M., Hobbs, M. and Egerton, J.R. TITLE Gene sequences and comparison of the fimbrial subunits representative of Bacteroides nodosus serotypes A to I: class I and class II strains JOURNAL Mol. Microbiol. 5 (3), 561-573 (1991) PUBMED 1675419 REFERENCE 35 AUTHORS Company, M., Arenas, J. and Abelson, J. TITLE Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes JOURNAL Nature 349 (6309), 487-493 (1991) PUBMED 1992352 REFERENCE 36 AUTHORS Umeda, M. and Ohtsubo, E. TITLE Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome JOURNAL Gene 98 (1), 1-5 (1991) PUBMED 1849492 REFERENCE 37 AUTHORS Hirvas, L., Koski, P. and Vaara, M. TITLE The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences JOURNAL J. Bacteriol. 173 (3), 1223-1229 (1991) PUBMED 1991717 REFERENCE 38 AUTHORS Bouvier, J. and Stragier, P. TITLE Nucleotide sequence of the lsp-dapB interval in Escherichia coli  JOURNAL Nucleic Acids Res. 19 (1), 180 (1991) PUBMED 2011499 REFERENCE 39 AUTHORS Dicker, I.B. and Seetharam, S. TITLE Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli JOURNAL J. Bacteriol. 173 (1), 334-344 (1991) PUBMED 1987124 REFERENCE 40 AUTHORS Grimm,B., Bull, A. and Breu, V. TITLE Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli JOURNAL Mol. Gen. Genet. 225 (1), 1-10 (1991) PUBMED 1900346 REFERENCE 41 AUTHORS Allen, B.L., Gerlach, G.F. and Clegg, S. TITLE Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae JOURNAL J. Bacteriol. 173 (2), 916-920 (1991) PUBMED 1670938 REFERENCE 42 AUTHORS Chen, H., Lawrence, C.B., Bryan, S.K. and Moses, R.E. TITLE Aphidicolin inhibits DNA polymerase II of Escherichia coli, an alpha-like DNA polymerase JOURNAL Nucleic Acids Res. 18 (23), 7185-7186 (1990) PUBMED 2124684 REFERENCE 43 AUTHORS Mallonee, D.H., White, W.B. and Hylemon, P.B. TITLE Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708 JOURNAL J. Bacteriol. 172 (12), 7011-7019 (1990) PUBMED 2254270 REFERENCE 44 AUTHORS Young, C., Collins-Emerson, J.M., Terzaghi, E.A. and Scott, D.B. TITLE Nucleotide sequence of Rhizobilm loti nodi JOURNAL Nucleic Acids Res. 18 (22), 6691 (1990) PUBMED 2251131 REFERENCE 45 AUTHORS Chen, H., Sun, Y., Stark, T., Beattil, W. and Moses, R.E. TITLE Nucleotide sequence and deletion analysis of the polB gene of Escherichia coli JOURNAL DNA Cell Biol. 9 (9), 631-635 (1990) PUBMED 2261080 REFERENCE 46 AUTHORS Eilani, G., Delarue, M., Poch, O., Gangloff, J. and Moras, D. TITLE Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs JOURNAL Nature 347 (6289), 203-206 (1990) PUBMED 2203971 REFERENCE 47 AUTHORS Showalter, R.E. and Silverman, M.R. TITLE Nucleotide sequence of a gene, hpt, for hypoxanthine phosphoribosyltransferase from Vibrio harveyi JOURNAL Nucleic Acids Res. 18 (15), 4621 (1990) PUBMED 2388850 REFERENCE 48 AUTHORS Martin-Verstraete, I., Debarbouille, M., Klier, A. and Rapoport, G. TITLE Levanase operon of Bacillus subtilis includes a fructose- specific phosphotransferase system regulating the expression of the operon JOURNAL J. Mol. Biol. 214 (3), 657-671 (1990) PUBMED 2117666 REFERENCE 49 AUTHORS Henrich, B., Monnerjahn, U. and Plapp, R. TITLE Peptidase D gene (pepD) of Escherichia coli K-12: nucleotide sequence, transcript mapping, and comparison with other peptidase genes JOURNAL J. Bacteriol. 172 (8), 4641-4651 (1990) PUBMED 1695895 REFERENCE 50 AUTHORS Nunn, D., Bergman, S. and Lory, S. TITLE Products of three accessory genes, pilB, pilC, and pilD, are required for biogenesis of Pseudomonas aeruginosa pili JOURNAL J. Bacteriol. 172 (6), 2911-2919 (1990) PUBMED 1971619 REFERENCE 51 AUTHORS Rosenthal, E.R. and Calyo, J.M. TITLE The nucleotide sequence of leuC from Salmonella typhimurium JOURNAL Nucleic Acids Res. 18 (10), 3072 (1990) PUBMED 2190189 REFERENCE 52 AUTHORS Kang, P.J. and Craig, E.A. TITLE Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation JOURNAL J. Bacteriol. 172 (4), 2055-2064 (1990) PUBMED 2180916 REFERENCE 53 AUTHORS Wurgler, S.M. and Richardson, C.C. TITLE Structure and regulation of the gene for dGTP triphosphohydrolase from Escherichia coli JOURNAL Proc. Natl. Acad. Sci. U.S.A. 87 (7), 2740-2744 (1990) PUBMED 2157212 REFERENCE 54 AUTHORS Schaaff, I., Hohmann, S. and Zimmermann, F.K. TITLE Molecular analysis of the structural gene for yeast transaldolase JOURNAL Eur. J. Biochem. 188 (3), 597-603 (1990) PUBMED 2185015 REFERENCE 55 AUTHORS Ricca, E. and Calyo, J.M. TITLE The nucleotide sequence of leuA from Salmonella typhimurium JOURNAL Nucleic Acids Res. 18 (5), 1290 (1990) PUBMED 2181403 REFERENCE 56 AUTHORS Honore, N. and Cole, S.T. TITLE Nucleotide sequence of the aroP gene encoding the general aromatic amino acid transport protein of Escherichia coli K-12: homology with yeast transport proteins JOURNAL Nucleic Acids Res. 18 (3), 653 (1990) PUBMED 2408019 REFERENCE 57 AUTHORS Angerer, A., Gaisser, S. and Braun, V. TITLE Nucleotide sequences of the sfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism JOURNAL J. Bacteriol. 172 (2), 572-578 (1990) PUBMED 2404942 REFERENCE 58 AUTHORS Surin, B.P., Watson, J.M., Hamilton, W.D., Economou, A. and Downie, J.A. TITLE Molecular characterization of the nodulation gene, nodT, from two biovars of Rhizobium leguminosarum JOURNAL Mol. Microbiol. 4 (2), 245-252 (1990) PUBMED 2338917 REFERENCE 59 AUTHORS Zhou, Z. and Syvanen, M. TITLE Identification and sequence of the drpA gene from Escherichia coli JOURNAL J. Bacteriol. 172 (1), 281-286 (1990) PUBMED 1688424 REFERENCE 60 AUTHORS Roncero, M.I., Jepsen, L.P., Stroman, P. and van Heeswijck, R. TITLE Characterization of a leuA gene and an ARS element from Mucor circinelloides JOURNAL Gene 84 (2), 335-343 (1989) PUBMED 2693214 REFERENCE 61 AUTHORS Ichikawa, S. and Kaji, A. TITLE Molecular cloning and expression of ribosome releasing factor JOURNAL J. Biol. Chem. 264 (33), 20054-20059 (1989) PUBMED 2684966 REFERENCE 62 AUTHORS Minami-Ishii, N., Taketani, S., Osumi, T. and Hashimoto, T. TITLE Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl-CoA hydratase. Structural and evolutionary relationships linked to the bifunctional enzyme of the peroxisomal beta-oxidation system JOURNAL Eur. J. Biochem. 185 (1), 73-78 (1989) PUBMED 2806264 REFERENCE 63 AUTHORS Matsubara, Y., Indo, Y., Naito, E., Ozasa, H., Glassberg, R., Vockley, J., Ikeda, Y., Kraus, J. and Tanaka, K. TITLE Molecular cloning and nucleotide sequence of cDNAs encoding the precursors of rat long chain acyl-coenzyme A, short chain acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases. Sequence homology of four enzymes of the acyl-CoA dehydrogenase family JOURNAL J. Biol. Chem. 264 (27), 16321-16331 (1989) PUBMED 2777793 REFERENCE 64 AUTHORS Roa, B.B., Connolly, D.M. and Winkler, M.E. TITLE Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG- apaH operon of Escherichia coli K-12 JOURNAL J. Bacteriol. 171 (9), 4767-4777 (1989) PUBMED 2670894 REFERENCE 65 AUTHORS Lindquist, S., Galleni, M., Lindberg, F. and Normark, S. TITLE Signalling proteins in enterobacterial AmpC beta-lactamase regulation JOURNAL Mol. Microbiol. 3 (8), 1091-1102 (1989) PUBMED 2691840 REFERENCE 66 AUTHORS Xie, Q.W., Tabor, C.W. and Tabor, H. TITLE Spermidine biosynthesis in Escherichia coli: promoter and termination regions of the speED operon JOURNAL J. Bacteriol. 171 (8), 4457-4465 (1989) PUBMED 2666401 REFERENCE 67 AUTHORS Sato, S., Nakada, Y. and Shiratsuchi, A. TITLE IS421, a new insertion sequence in Escherichia coli JOURNAL FEBS Lett. 249 (1), 21-26 (1989) PUBMED 2542093 REFERENCE 68 AUTHORS Liu, J.D. and Parkinson, J.S. TITLE Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control JOURNAL J. Bacteriol. 171 (3), 1254-1261 (1989) PUBMED 2537812 REFERENCE 69 AUTHORS Henrich, B., Schroeder, U., Frank, R.W. and Plapp, R. TITLE Accurate mapping of the Escherichia coli pepD gene by sequence analysis of its 5′ flanking region JOURNAL Mol. Gen. Genet. 215 (3), 369-373 (1989) PUBMED 2651887 REFERENCE 70 AUTHORS Lipinska, B., Sharma, S. and Georgopoulos, C. TITLE Sequence analysis and regulation of the htrA gene of Escherichia coli: a sigma 32-Independent mechanism of heat-inducible transcription JOURNAL Nucleic Acids Res. 16 (21), 10053-10067 (1988) PUBMED 3057437 REFERENCE 71 AUTHORS Sung, Y.C. and Fuchs, J.A. TITLE Characterization of the cyn operon in Escherichia coli K12 JOURNAL J. Bio. Chem. 263 (29), 14769-14775 (1988) PUBMED 3049588 REFERENCE 72 AUTHORS Lozoya, E., Hoffmann, H., Douglas, C., Schulz, W., Scheel, D. and Hahlbrock, K. TITLE Primary structures and catalytic properties of isoenzymes encoded by the two 4-coumarate: CoA ligase genes in parsley JOURNAL Eur. J. Biochem. 176 (3), 661-667 (1988) PUBMED 3169018 REFERENCE 73 AUTHORS Andrews, S.C. and Guest, J.R. TITLE Nucleotide sequence of the gene encoding the GMP reductase of Escherichia coli K12 JOURNAL Biochem. J. 255 (1), 35-43 (1988) PUBMED 2904262 REFERENCE 74 AUTHORS Jaiswal, A.K., McBride, O.W., Adesnik, M. and Nebert, D.W. TITLE Human dioxin-inducible cytosolic NAD(P)H:menadione oxidoreductase. cDNA sequence and localization of gene to chromosome 16 JOURNAL J. Bio. Chem. 263 (27), 13572-13578 (1988) PUBMED 2843525 REFERENCE 75 AUTHORS Karpel, R., Olami, Y., Taglicht, D., Schuldiner, S. and Padan, E. TITLE Sequencing of the gene ant which affects the Na+/H+ antiporter activity in Escherichia coli JOURNAL J. Bio. Chem. 263 (21), 10408-10414 (1988) PUBMED 2839489 REFERENCE 76 AUTHORS Mellano, M.A. and Cooksey, D.A. TITLE Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato JOURNAL J. Bacteriol. 170 (6), 2879-2883 (1988) PUBMED 3372485 REFERENCE 77 AUTHORS Coleman, J. and Raetz, C.R. TITLE First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene JOURNAL J. Bacteriol. 170 (3), 1268-1274 (1988) PUBMED 3277952 REFERENCE 78 AUTHORS Gebhard, W., Schreitmuller, T., Hochstrasser, K. and Wachter, E. TITLE Complementary DNA and derived amino acid sequence of the precursor of one of the three protein components of the inter-alpha- trypsin inhibitor complex JOURNAL FEBS Lett. 229 (1), 63-67 (1988) PUBMED 2450046 REFERENCE 79 AUTHORS Tomasiewicz, H.G. and McHenry, C.S. TITLE Sequence analysis of the Escherichia coli dnaE gene JOURNAL J. Bacteriol. 169 (12), 5735-5744 (1987) PUBMED 3316192 REFERENCE 80 AUTHORS Crowell, D.N., Reznikoff, W.S. and Raetz, C.R. TITLE Nucleotide sequence of the Escherichia coli gene for lipid A disaccharide synthase JOURNAL J. Bacteriol. 169 (12), 5727-5734 (1987) PUBMED 2824445 REFERENCE 81 AUTHORS Tabor, C.W. and Tabor, H. TITLE The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of 5-adenosylmethionine decarboxylase JOURNAL J. Biol. Chem. 262 (33), 16037-16040 (1987) PUBMED 3316212 REFERENCE 82 AUTHORS Nonet, M.L., Marvel, C.C. and Tolan, D.R. TITLE The hisT-purF region of the Escherichia coli K-12 chromosome. Identification of additional genes of the hisT and purF operons JOURNAL J. Biol. Chem. 262 (25), 12209-12217 (1987) PUBMED 3040734 REFERENCE 83 AUTHORS Coulton, J.W., Mason, P. and Allatt, D.D. TITLE fhuC and fhuD genes for iron (III)-ferrichrome transport into Escherichia coli K-12 JOURNAL J. Bacteriol. 169 (8), 3844-3849 (1987) PUBMED 3301821 REFERENCE 84 AUTHORS Horiuchi, T., Nagasawa, T., Takano, K. and Sekiguchi, M. TITLE A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12 JOURNAL Mol. Gen. Genet. 206 (2), 356-357 (1987) PUBMED 3295485 REFERENCE 85 AUTHORS Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A. and Chang, S. TITLE Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure JOURNAL J. Bacteriol. 169 (2), 751-757 (1987) PUBMED 3027045 REFERENCE 86 AUTHORS Gronger, P., Manian, S.S., Reilander, H., O'Connell, M., Priefer, U.B. and Puhler, A. TITLE Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame JOURNAL Nucleic Acids Res. 15 (1), 31-49 (1987) PUBMED 3029674 REFERENCE 87 AUTHORS Richardson, K.K., Richardson, F.C., Crosby, R.M., Swenberg, J.A. and Skopek, T.R. TITLE DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N- nitrosourea JOURNAL Proc. Natl. Acad. Sci. U.S.A. 84 (2), 344-348 (1987) PUBMED 3540961 REFERENCE 88 AUTHORS Chye, M.L. and Pittard, J. TITLE Transcription control of the aroP gene in Escherichia coli K- 12: analysis of operator mutants JOURNAL J. Bacteriol. 169 (1), 386-393 (1987) PUBMED 3025182 REFERENCE 89 AUTHORS Blanchin-Roland, S., Blanquet, S., Schmitter, J.M. and Fayat, G. TITLE The gene for Escherichia coli diadensoine tetraphosphatase is located immediately clockwise to folA and forms an operon with ksgA JOURNAL Mol. Gen. Genet. 205 (3), 515-522 (1986) PUBMED 3031429 REFERENCE 90 AUTHORS Takano, K., Nakabeppu, Y., Maki, H., Horiuchi, T. and Sekiguchi, M. TITLE Structure and function of dnaQ and mutD mutators of Escherichia coli JOURNAL Mol. Gen. Genet. 205 (1), 9-13 (1986) PUBMED 3540531 REFERENCE 91 AUTHORS Mackie, G.A. TITLE Structure of the DNA distal to the gene for ribosomal protein S20 in Escherichia coli K12: presence of a strong terminator and an IS1 element JOURNAL Nucleic Acids Res. 14 (17), 6965-6981 (1986) PUBMED 2429258 REFERENCE 92 AUTHORS Koster, W. and Braun, V. TITLE Iron hydroxamate transport of Escherichia coli: nucleotide sequence of the fhuB gene and identification of the protein JOURNAL Mol. Gen. Genet. 204 (3), 435-442 (1986) PUBMED 3020380 REFERENCE 93 AUTHORS Breton, R., Sanfacon, H., Papayannopoulos, I., Biemann, K. and Lapointe, J. TITLE Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl- tRNA synthetases JOURNAL J. Biol. Chem. 261 (23), 10610-10617 (1986) PUBMED 3015933 REFERENCE 94 AUTHORS Birnbaum, M.J., Haspel, H.C. and Rosen, O.M. TITLE Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein JOURNAL Proc. Natl. Acad. Sci. U.S.A. 83 (16), 5784-5788 (1986) PUBMED 3016720 REFERENCE 95 AUTHORS Cox, E.C. and Horner, D.L. TITLE DNA sequence and coding properties of mutD(dnaQ) a dominant Escherichia coli mutator gene JOURNAL J. Mol. Biol. 190 (1), 113-117 (1986) PUBMED 3023634 REFERENCE 96 AUTHORS Ohki, M., Tamura, F., Nishimura, S. and Uchida, H. TITLE Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product JOURNAL J. Biol. Chem. 261 (4), 1778-1781 (1986) PUBMED 3003084 REFERENCE 97 AUTHORS Coulton, J.W., Mason, P., Cameron, D.R., Carmel, G., Jean, R. and Rode, H.N. TITLE Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12 JOURNAL J. Bacteriol. 165 (1), 181-192 (1986) PUBMED 3079747 REFERENCE 98 AUTHORS Lee, N., Gielow, W., Martin, R., Hamilton, E. and Fowler, A. TITLE The organization of the araBAD operon of Escherichia coli JOURNAL Gene 47 (2-3), 231-244 (1986) PUBMED 3549454 REFERENCE 99 AUTHORS Sekiguchi, T., Ortega-Cesena, J., Nosoh, Y., Ohashi, S., Tsuda, K. and Kanaya, S. TITLE DNA and amino-acid sequences of 3-isopropylmalate dehydrogenase of Bacillus coagulans. Comparison with the enzymes of Saccharomyces cerevisiae and Thermus thermophilus JOURNAL Biochim. Biophys. Acta 867, 36-44 (1986) REFERENCE 100 AUTHORS Chong, P., Hui, I., Loo, T. and Gillam, S. TITLE Structural analysis of a new GC-specific insertion element IS186 JOURNAL FEBS Lett. 192 (1), 47-52 (1985) PUBMED 2996940 REFERENCE 101 AUTHORS Icho, T., Sparrow, C.P. and Raetz, C.R. TITLE Molecular cloning and sequencing of the gene for CDP- diglyceride synthetase of Escherichia coli JOURNAL J. Biol. Chem. 260 (22), 12078-12083 (1985) PUBMED 2995358 REFERENCE 102 AUTHORS Nomura, T., Aiba, H. and Ishihama, A. TITLE Transcriptional organization of the convergent overlapping dnaQ-rnh genes of Escherichia coli JOURNAL J. Biol. Chem. 260 (11), 7122-7125 (1985) PUBMED 2987244 REFERENCE 103 AUTHORS Kamio, Y., Lin, C.K., Regue, M. and Wu, H.C. TITLE Characterization of the ileS-lsp operon in Escherichia coli. Identification of an open reading frame upstream of the ileS gene and potential promoter(s) for the ileS-lsp operon JOURNAL J. Biol. Chem. 260 (9), 5616-5620 (1985) PUBMED 2985604 REFERENCE 104 AUTHORS Cowing, D.W., Bardwell, J.C., Craig, E.A., Woolford, C., Hendrix, R.W. and Gross, C.A. TITLE Consensus sequence for Escherichia coli heat shock gene promoters JOURNAL Proc. Natl. Acad. Sci. U.S.A. 82 (9), 2679-2683 (1985) PUBMED 3887408 REFERENCE 105 AUTHORS Broome-Smith, J.K., Edelman, A., Yousif, S. and Spratt, B.G. TITLE The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12 JOURNAL Eur. J. Biochem. 147 (2), 437-446 (1985) PUBMED 3882429 REFERENCE 106 AUTHORS Becerril, B., Valle, F., Merino, E., Riba, L. and Bolivar, F. TITLE Repetitive extragenic palindromic (REP) sequences in the Escherichia coli gdhA gene JOURNAL Gene 37 (1-3), 53-62 (1985) PUBMED 3902576 REFERENCE 107 AUTHORS Friedberg, D., Rosenthal, E.R., Jones, J.W. and Calvo, J.M. TITLE Characterization of the 3′ end of the leucine operon of Salmonella typhimurium JOURNAL Mol. Gen. Genet. 199 (3), 486-494 (1985) PUBMED 2993799 REFERENCE 108 AUTHORS Bouvier, J., Richaud, C., Richaud, F., Patte, J.C. and Stragier, P. TITLE Nucleotide sequence and expression of the Escherichia coli dapB gene JOURNAL J. Biol. Chem. 259 (23), 14829-14834 (1984) PUBMED 6094578 REFERENCE 109 AUTHORS Richaud, C., Richaud, F., Martin, C., Haziza, C. and Patte, J.C. TITLE Regulation of expression and nucleotide sequence of the Escherichia coli dapD gene JOURNAL J. Biol. Chem. 259 (23), 14824-14828 (1984) PUBMED 6094577 REFERENCE 110 AUTHORS Nuesch, J. and Schumperli, D. TITLE Structural and functional organization of the gpt gene region of Escherichia coli JOURNAL Gene 32 (1-2), 243-249 (1984) PUBMED 6397401 REFERENCE 111 AUTHORS Jagadeeswaran, P., Ashman, C.R., Roberts, S. and Langenberg, J. TITLE Nucleotide sequence and analysis of deletion mutants of the Escherichia coli gpt gene in plasmid pSV2 gpt JOURNAL Gene 31 (1-3), 309-313 (1984) PUBMED 6396164 REFERENCE 112 AUTHORS Deutch, A.H., Rushlow, K.E. and Smith, C.J. TITLE Analysis of the Escherichia coli proBA locus by DNA and protein sequencing JOURNAL Nucleic Acids Res. 12 (15), 6337-6355 (1984) PUBMED 6089111 REFERENCE 113 AUTHORS Bouvier, J., Patte, J.C. and Stragier, P. TITLE Multiple regulatory signals in the control region of the Escherichia coli carAB operon JOURNAL Proc. Natl. Acad. Sci. U.S.A. 81 (13), 4139-4143 (1984) PUBMED 6377309 REFERENCE 114 AUTHORS Innis, M.A., Tokunaga, M., WIlliams, M.E., Loranger, J.M., Chang, S.Y., Chang, S. and Wu, H.C. TITLE Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene JOURNAL Proc. Natl. Acad. Sci. U.S.A. 81 (12), 3708-3712 (1984) PUBMED 6374664 REFERENCE 115 AUTHORS Bardwell, J.C. and Craig, E.A. TITLE Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous JOURNAL Proc. Natl. Acad. Sci. U.S.A. 81 (3), 848-852 (1984) PUBMED 6322174 REFERENCE 116 AUTHORS Pratt, D. and Subramani, S. TITLE Nucleotide sequence of the Escherichia coli xanthine-guanine phosphoribosyl transferase gene JOURNAL Nucleic Acids Res. 11 (24), 8817-8823 (1983) PUBMED 6324103 REFERENCE 117 AUTHORS Richardson, K.K., Fostel, J. and Skopek, T.R. TITLE Nucleotide sequence of the xanthine guanine phosphoribosyl transferase gene of E. coli JOURNAL Nucleic Acids Res. 11 (24), 8809-8816 (1983) PUBMED 6324102 REFERENCE 118 AUTHORS Parsot, C., Cossart, P., Saint-Girons, I. and Cohen, G.N. TITLE Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12 JOURNAL Nucleic Acids Res. 11 (21), 7331-7345 (1983) PUBMED 6316258 REFERENCE 119 AUTHORS Stephens, P.E., Lewis, H.M., Darlison, M.G. and Guest, J.R. TITLE Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12 JOURNAL Eur. J. Biochem. 135 (3), 519-527 (1983) PUBMED 6352260 REFERENCE 120 AUTHORS Stephens, P.E., Darlison, M.G., Lewis, H.M. and Guest, J.R. TITLE The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component JOURNAL Eur. J. Biochem. 133 (3), 481-489 (1983) PUBMED 6345153 REFERENCE 121 AUTHORS Stephens, P.E., Darlison, M.G., Lewis, H.M. and Guest, J.R. TITLE The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component JOURNAL Eur. J. Biochem. 133 (1), 155-162 (1983) PUBMED 6343085 REFERENCE 122 AUTHORS Kanaya, S. and Crouch, R.J. TITLE Low levels of RNase H activity in Escherichia coli FB2 rnh result from a single-base change in the structural gene of RNase H JOURNAL J. Bacteriol. 154 (2), 1021-1026 (1983) PUBMED 6302075 REFERENCE 123 AUTHORS Overbeeke, N., Bergmans, H., van Mansfeld, F. and Lugtenberg, B. TITLE Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 JOURNAL J. Mol. Biol. 163 (4), 513-532 (1983) PUBMED 6341601 REFERENCE 124 AUTHORS Gilson, E., Nikaido, H. and Hofnung, M. TITLE Sequence of the malK gene in E.coli K12 JOURNAL Nucleic Acids Res. 10 (22), 7449-7458 (1982) PUBMED 6296778 REFERENCE 125 AUTHORS Stoner, C.M. and Schleif, R. TITLE Is the amino acid but not the nucleotide sequence of the Escherichia coli araC gene conserved? JOURNAL J. Mol. Biol. 154 (4), 649-652 (1982) PUBMED 6283093 REFERENCE 126 AUTHORS An, G., Bendiak, D.S., Mamelak, L.A. and Friesen, J.D. TITLE Organization and nucleotide sequence of a new ribosomal operon in Escherichia coli containing the genes for ribosomal protein S2 and elongation factor Ts JOURNAL Nucleic Acids Res. 9 (16), 4163-4172 (1981) PUBMED 6272196 REFERENCE 127 AUTHORS Mackie, G.A. TITLE Nucleotide sequence of the gene for ribosomal protein S20 and its flanking regions JOURNAL J. Biol. Chem. 256 (15), 8177-8182 (1981) PUBMED 6267039 REFERENCE 128 AUTHORS Little, J.W., Mount, D.W. and Yanisch-Perron, C.R. TITLE Purified lexA protein is a repressor of the recA and lexA genes JOURNAL Proc. Natl. Acad. Sci. U.S.A. 78 (7), 4199-4203 (1981) PUBMED 7027255 REFERENCE 129 AUTHORS Mulligan, R.C. and Berg, P. TITLE Factors governing the expression of a bacterial gene in mammalian cells JOURNAL Mol. Cell. Biol. 1 (5), 449-459 (1981) PUBMED 6100966 REFERENCE 130 AUTHORS Lee, N.L., Gielow, W.O. and Wallace, R.G. TITLE Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli JOURNAL Proc. Natl. Acad. Sci. U.S.A. 78 (2), 752-756 (1981) PUBMED 6262769 REFERENCE 131 AUTHORS Cossart, P., Katinka, M. and Yaniv, M. TITLE Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions JOURNAL Nucleic Acids Res. 9 (2), 339-347 (1981) PUBMED 6259626 REFERENCE 132 AUTHORS Miyada, C.G., Horwitz, A.H., Cass, L.G., Timko, J. and Wilcox, G. TITLE DNA sequence of the araC regulatory gene from Escherichia coli B/r JOURNAL Nucleic Acids Res. 8 (22), 5267-5274 (1980) PUBMED 7008027 REFERENCE 133 AUTHORS Katinka, M., Cossart, P., Sibilli, L., Saint-Girons, I., Chalyignac, M.A., Le Bras, G., Cohen, G.N. and Yaniy, M. TITLE Nucleotide sequence of the thrA gene of Escherichia coli JOURNAL Proc. Natl. Acad. Sci. U.S.A. 77 (10), 5730-5733 (1980) PUBMED 7003595 REFERENCE 134 AUTHORS Ogden, S., Haggerty, D., Stoner, C.M., Kolodrubetz, D. and Schleif, R. TITLE The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation JOURNAL Proc. Natl. Acad. Sci. U.S.A. 77 (6), 3346-3350 (1980) PUBMED 6251457 REFERENCE 135 AUTHORS Smith, D.R. and Calyo, J.M. TITLE Nucleotide sequence of the E coli gene coding for dihydrofolate reductase JOURNAL Nucleic Acids Res. 8 (10), 2255-2274 (1980) PUBMED 6159575 REFERENCE 136 AUTHORS Johnsrud, L. TITLE DNA sequence of the transposable element IS1 JOURNAL Mol. Gen. Genet. 169 (2), 213-218 (1979) PUBMED 375010 REFERENCE 137 AUTHORS Smith, B.R. and Schleif, R. TITLE Nucleotide sequence of the L-arabinose regulatory region of Escherichia coli K12 JOURNAL J. Biol. Chem. 253 (19), 6931-6933 (1978) PUBMED 357433 REFERENCE 138 AUTHORS Greenfield, L., Boone, T. and Wilcox, G. TITLE DNA sequence of the araBAD promoter in Escherichia coli B/r JOURNAL Proc. Natl. Acad. Sci. U.S.A. 75 (10), 4724-4728 (1978) PUBMED 368797 REFERENCE 139 AUTHORS Young, R.A. and Steitz, J.A. TITLE Complementary sequences 1700 nucleotides apart form a ribonuclease III cleavage site in Escherichia coli ribosomal precursor RNA JOURNAL Proc. Natl. Acad. Sci. U.S.A. 75 (8), 3593-3597 (1978) PUBMED 358189 REFERENCE 140 AUTHORS Ohtsubo, H. and Ohtsubo, E. TITLE Nucleotide sequence of an insertion element, IS1 JOURNAL Proc. Natl. Acad. Sci. U.S.A. 75 (2), 615-619 (1978) PUBMED 273224 REFERENCE 141 AUTHORS Musso, R., Di Lauro, R., Rosenberg, M. and de Crombrugghe, B. TITLE Nucleotide sequence of the operator-promoter region of the galactose operon of Escherichia coli JOURNAL Proc. Natl. Acad. Sci. U.S.A. 74 (1), 106-110 (1977) PUBMED 319453 REFERENCE 142 (bases 1 to 4646332) CONSRTM NCBI Genome Project TITLE Direct Submission JOURNAL Submitted (10-NOV-2005) National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA REFERENCE 143 (bases 1 to 4646332) AUTHORS Mori, H., Horiuchi, T. and Hirai, A. TITLE Direct Submission JOURNAL Submitted (22-AUG-2005) Hirotada Mori, Graduate School of Biological Sciences, Nara Institute of Science and Technology; 8916-5 Takayama, Ikoma, Nara 630-0101, Japan (E-mail:hmori@gtc.naist.jp, Tel: 81-743-72-5660, Fax: 81-743-72- 5669) COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from AP009048. COMPLETENESS: full length. FEATURES Location/Qualifiers source complement(<1..<5861) /organism = ″Escherichia coli str. K-12 substr. W3110″ /mol_type = ″genomic DNA″ /strain = ″K-12″ /sub_strain = ″W3110″ /db_xref = ″taxon:316407″ gene complement(<1..6) /gene = ″dcuD″ CDS complement(<1..6) /gene = ″dcuD″ /note = ″ECK3216:JW3196:b3227″ /codon_start = 1 /transl_table = 11 /product = ″predIcted transporter″ /protein_id = ″AP_003769.1″ /db_xref = ″GI:89109989″ /translation = ″MFGIIISVIVLITMGYLILKNYKPQVVLAAAGIFLMMCGVWLGF GGVLDPTKSSGYLIVDIYNEILRMLSNRIAGLGLSIMAVGGYARYMERIGASRAMVSL LSRPLKLIRSPYIILSATYVIGQIMAQFITSASGLGMLLMVTLFPTLVSLGVSRLSAV AVIATTMSIEWGILETNSIFAAQVAGMKIATYFFHYQLPVASCVIISVAISHFFVQRA FDKKDKNINHEQAEQKALDNVPPLYYAILPVMPLILMLGSLFLAHVGLMQSELHLVVV MLLSLTVTMFVEFFRKHNLRETMDDVQAFFDGMGTQFANVVTLVVAGEIFAKGLTTIG TVDAVIRGAEHSGLGGIGVMIIMALVIAICAIVMGSGNAPFMSFASLIPNIAAGLHVP AVVMIMPMHFATTLARAVSPITAVVVVTSGIAGVSPFAVVKRTAIPMAVGFVVNMIAT ITLFY″ (SEQ ID NO: 35) primer 330..348 /label = ″ck nanR3 control primer″ gene 386..1177 /gene = ″nanR″ CDS 386..1177 /gene = ″nanR″ /note = ″ECK3215:JW3195:b3226″ /codon_start = 1 /transl_table = 11 /product = ″DNA-binding transcriptional dual regulator″ /protein_id = ″AP_003768.1″ /db_xref = ″GI:89109988″ /translation = ″MGLMNAFDSQTEDSSPAIGRNLRSRPLARKKLSEMVEEELEQMI RRREFGEGEQLPSERELMAFFNVGRPSVREALAALKRKGLVQINNGERARVSRPSADT IIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQ SLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDQALHEHNNVS YQQHIAIVDAIRRHDPDEADRALQSHLNSVSATWHAFGQTTNKKK″ (SEQ ID NO: 36) primer 1005..1025 /label = ″nanR ck2 control primer″ primer 1126..1146 /label = ″nanAFck control primer″ promoter 1178..1278 /label = ″nan operon promoter region″ Site 1187..1191 /site_type = ″binding site″ /label = ″CAP binding″ Site 1198..1202 /site_type = ″binding site″ /label = ″CAP binding″ promoter 1241..1246 /label = -10 primer_bind 1252..1301 /note = ″for dnanA:: or dnanATE::scar deletions″ /label = ″H1-dnanA lambda red primer″ mRNA 1255 /label = +1 mRNA 1267 /label = +13 mRNA 1279 /label = +25 gene 1299..2192 /gene = ″nanA″ CDS 1299..2192 /gene = ″nanA″ /note = ″ECK3214:JW3194:b3225″ /codon_start = 1 /transl_table = 11 /product = ″N-acetylneuraminate lyase″ /protein_Id = ″AP_003767.1″ /db_xref = ″GI:89109987″ /translation = ″MATNLRGVMAALLTPFDQQQALDKASLRRLVQFNIQQGIDGLYV GGSTGEAFVQSLSEREQVLEIVAEEAKGKIKLIAHVGCVSTAESQQLAASAKRYGFDA VSAVTPFYYPFSFEEHCDHYRAIIDSADGLPMVVYNIPALSGVKLTLDQINTLVTLPG VGALKQTSGDLYQMEQIRREHPDLVLYNGYDEIFASGLLAGADGGIGSTYNIMGWRYQ GIVKALKEGDIQTAQKLQTECNKVIDLLIKTGVFRGLKTVLHYMDVVSVPLCRKPFGP VDEKYLPELKALAQQLMQERG″ (SEQ ID NO: 37) Region 1302..4424 /label = ″DELETION nanATE″ primer_bind complement(2175..2224) /label = ″H2-dnanA lambda red primer″ gene 2301..3791 /gene = ″nanT″ CDS 2301..3791 /gene = ″nanT″ /note = ″ECK3213:JW3193:b3224″ /codon_start = 1 /transl_table = 11 /product = ″sialic acid transporter″ /protein_Id = ″AP_003766.1″ /db_xref = ″GI:89109986″ /translation = ″MSTTTQNIPWYRHLNRAQWRAFSAAWLGYLLDGFDFVLIALVLT EVQGEFGLTTVQAASLISAAFISRWFGGLMLGAMGDRYGRRLAMVTSIVLFSAGTLAC GFAPGYITMFIARLVIGMGMAGEYGSSATYVIESWPKHLRNKASGFLISGFSVGAVVA AQVYSLVVPVWGWRALFFIGILPIIFALWLRKNIPEAEDWKEKHAGKAPVRTMVDILY RGEHRIANIVMTLAAATALWFCFAGNLQNAAIVAVLGLLCAAIFISFMVQSAGKRWPT GVMLMVVVLFAFLYSWPIQALLPTYLKTDLAYNPHTVANVLFFSGFGAAVGCCVGGFL GDWLGTRKAYVCSLLASQLLIIPVFAIGGANVWVLGLLLFFQQMLGQGIAGILPKLIG GYFDTDQRAAGLGFTYNVGALGGALAPIIGALIAQRLDLGTALASLSFSLTFVVILLI GLDMPSRVQRWLRPEALRTHDAIDGKPFSGAVPFGSAKNDLVKTKS″ (SEQ ID NO: 38) primer complement(2329..2350) /label = ″nanARck control primer″ primer_bind 3792..3841 /label = ″H1-dnanE lambda red primer″ gene 3839..4528 /gene = ″nanE″ CDS 3839..4528 /gene = ″nanE″ /note = ″ECK3212:JW3192:b3223″ /codon_start = 1 /transl_table = 11 /product = ″predicted N-acetylmannosamine-6-Pepimerase″ /protein_id = ″AP_003765.1″ /db_xref = ″GI:89109985″ /translation = ″MSLLAQLDQKIAANGGLIVSCQPVPDSPLDKPEIVAAMALAAEQ AGAVAIRIEGVANLQATRAVVSVPIIGIVKRDLEDSPVRITAYIEDVDALAQAGADII AIDGTDRPRPVPVETLLARIHHHGLLAMTDCSTPEDGLACQKLGAETIGTTLSGYTTP ETPEEPDLALVKTLSDAGCRVIAEGRYNTPAQAADAMRHGAWAVTVGSAITRLEHICQ WYNTAMKKAVL″ (SEQ ID NO: 39) primer_bind complement(4425..4474) /note = ″for dnanATE::scar deletion″ /label = ″H2-dnanE lambda red primer″ RBS 4425..4448 /label = ″C-terminal gibberish peptide fused to KD13 scar peptide″ RBS 4449..4451 /label = ″NEW STOP gibberish peptide after resolution of cassette″ primer_bind 4486..4530 /label = ″nanK-H1 lambda red primer″ RBS 4515..4520 /label = ″nanK RBS″ gene 4525..5400 /gene = ″nanK″ CDS 4525..5400 /gene = ″nanK″ /note = ″ECK3211:JW5538:b3222″ /codon_start = 1 /transl_table = 11 /product = ″predicted N-acetylmannosamine kinase″ /protein_id = ″AP_003764.1″ /db_xref = ″GI:89109984″ /translation = ″MTTLAIDIGGTKLAAALIGADGQIRDRRELPTPASQTPEALRDA LSALVSPLQAHAQRVAIASTGIIRDGSLLALNPHNLGGLLHFPLVKTLEQLTNLPTIA INDAQAAAWAEFQALDGDITDMVFITVSTGVGGGVVSGCKLLTGPGGLAGHIGHTLAD PHGPVCGCGRTGCVEATASGRGIAAAAQGELAGADAKTIFTRAGQGDEQAQQLIHRSA RTLARLIADIKATTDCQCVVVGGSVGLAEGYLALVETYLAQEPAAFHVDLLAAHYRHD AGLLGAALLAQGEKL″ (SEQ ID NO: 40 RBS 4526..4528 /label = ″Native Stop for NanE″ primer complement(5065..5083) /label = ″nanKck1 control primer″ primer_bind complement(5380..5424) /label = ″nanK-H2 lambda red primer″ gene 5397..5861 /gene = ″yhcH″ CDS 5397..5861 /gene = ″yhcH″ /note = ″ECK3210:JW3190:b3221″ /codon_start = 1 /transl_table = 11 /product = ″hypothetical protein″ /protein_id = ″AP_003763.1″ /db_xref = ″GI:89109983″ /translation = ″MMMGEVQSLPSAGLHPALQDALTLALAARPQEKAPGRYELQGDN IFMNVMTFNTQSPVEKKAELHEQYIDIQLLLNGEERILFGMAGTARQCEEFHHEDDYQ LCSTIDNEQAIILKPGMFAVFMPGEPHKPGCVVGEPGEIKKVVVKVKADLMA″ (SEQ ID NO: 41) ORIGIN    1 GAACATTGTT GAACTCCGTG TCAAAAGAAA ACGGTCAATC CCATAAACGG CAGATTGAAA   61 ACAACGATGT TATATTTTTT GCAAGGCTAT TTATGGTGCG GATGTCGTGT TTTTAATTGT  121 AGGTGAGGTG ATTTTTCATT AAAAAATATG CGCTTATGAT TATTTTGTAA GAACACATTC  181 ATAATATTCA TAATGCTCGT GAATAGTCTT ATAAATAATT CAAACGGGAT GTTTTTATCT  241 GCGTTACATT AATTTTTCGC AATAGTTAAT TATTCCGTTA ATTATGGTAA TGATGAGGCA  301 CAAAGAGAAA ACCCTGCCAT TTTCCCCTAC TTTCAATCCT GTGATAGGAT GTCACTGATG  361 ATGTTAATCA CACTGACCTT ACAGAATGGG CCTTATGAAC GCATTTGATT CGCAAACCGA  421 AGATTCTTCA CCTGCAATTG GTCGCAACTT GCGTAGCCGC CCGCTGGCGC GTAAAAAACT  481 CTCCGAAATG GTGGAAGAAG AGCTGGAACA GATGATCCGC CGTCGTGAAT TTGGCGAAGG  541 TGAACAATTA CCGTCTGAAC GCGAACTGAT GGCGTTCTTT AACGTCGGGC GTCCTTCGGT  601 GCGTGAAGCG CTGGCAGCGT TAAAACGCAA AGGTCTGGTG CAAATAAACA ACGGCGAACG  661 CGCTCGCGTC TCGCGTCCTT CTGCGGACAC TATCATCGGT GAGCTTTCCG GCATGGCGAA  721 AGATTTCCTT TCTCATCCCG GTGGGATTGC CCATTTCGAA CAATTACGTC TGTTCTTTGA  781 ATCCAGTCTG GTGCGCTATG CGGCTGAACA TGCCACCGAT GAGCAAATCG ATTTGCTGGC  841 AAAAGCACTG GAAATCAACA GTCAGTCGCT GGATAACAAC GCGGCATTCA TTCGTTCAGA  901 CGTTGATTTC CACCGCGTGC TGGCGGAGAT CCCCGGTAAC CCAATCTTCA TGGCGATCCA  961 CGTTGCCCTG CTCGACTGGC TTATTGCCGC ACGCCCAACG GTTACCGATC AGGCACTGCA 1021 CGAACATAAC AACGTTAGTT ATCAACAGCA TATTGCGATC GTTGATGCGA TCCGCCGTCA 1081 TGATCCTGAC GAAGCCGATC GTGCGTTGCA ATCGCATCTC AACAGCGTCT CTGCTACCTG 1141 GCACGCTTTC GGTCAGACCA CCAACAAAAA GAAATAATGC CACTTTAGTG AAGCAGATCG 1201 CATTATAAGC TTTCTGTATG GGGTGTTGCT TAATTGATCT GGTATAACAG GTATAAAGGT 1261 ATATCGTTTA TCAGACAAGC ATCACTTCAG AGGTATTTAT GGCAACGAAT TTACGTGGCG 1321 TAATGGCTGC ACTCCTGACT CCTTTTGACC AACAACAAGC ACTGGATAAA GCGAGTCTGC 1381 GTCGCCTGGT TCAGTTCAAT ATTCAGCAGG GCATCGACGG TTTATACGTG GGTGGTTCGA 1441 CCGGCGAGGC CTTTGTACAA AGCCTTTCCG AGCGTGAACA GGTACTGGAA ATCGTCGCCG 1501 AAGAGGCGAA AGGTAAGATT AAACTCATCG CCCACGTCGG TTGCGTCAGC ACCGCCGAAA 1561 GCCAACAACT TGCGGCATCG GCTAAACGTT ATGGCTTCGA TGCCGTCTCC GCCGTCACGC 1621 CGTTCTACTA TCCTTTCAGC TTTGAAGAAC ACTGCGATCA CTATCGGGCA ATTATTGATT 1681 CGGCGGATGG TTTGCCGATG GTGGTGTACA ACATTCCAGC CCTGAGTGGG GTAAAACTGA 1741 CCCTGGATCA GATCAACACA CTTGTTACAT TGCCTGGCGT AGGTGCGCTG AAACAGACCT 1801 CTGGCGATCT CTATCAGATG GAGCAGATCC GTCGTGAACA TCCTGATCTT GTGCTCTATA 1861 ACGGTTACGA CGAAATCTTC GCCTCTGGTC TGCTGGCGGG CGCTGATGGT GGTATCGGCA 1921 GTACCTACAA CATCATGGGC TGGCGCTATC AGGGGATCGT TAAGGCGCTG AAAGAAGGCG 1981 ATATCCAGAC CGCGCAGAAA CTGCAAACTG AATGCAATAA AGTCATTGAT TTACTGATCA 2041 AAACGGGCGT ATTCCGCGGC CTGAAAACTG TCCTCCATTA TATGGATGTC GTTTCTGTGC 2101 CGCTGTGCCG CAAACCGTTT GGACCGGTAG ATGAAAAATA TCTGCCAGAA CTGAAGGCGC 2161 TGGCCCAGCA GTTGATGCAA GAGCGCGGGT GAGTTGTTTC CCCTCGCTCG CCCCTACCGG 2221 GTGAGGGGAA ATAAACGCAT CTGTACCCTA CAATTTTCAT ACCAAAGCGT GTGGGCATCG 2281 CCCACCGCGG GAGACTCACA ATGAGTACTA CAACCCAGAA TATCCCGTGG TATCGCCATC 2341 TCAACCGTGC ACAATGGCGC GCATTTTCCG CTGCCTGGTT GGGATATCTG CTTGACGGTT 2401 TTGATTTCGT TTTAATCGCC CTGGTACTCA CCGAAGTACA AGGTGAATTC GGGCTGACGA 2461 CGGTGCAGGC GGCAAGTCTG ATCTCTGCAG CCTTTATCTC TCGCTGGTTC GGCGGCCTGA 2521 TGCTCGGCGC TATGGGTGAC CGCTACGGGC GTCGTCTGGC AATGGTCACC AGCATCGTTC 2581 TCTTCTCGGC CGGGACGCTG GCCTGCGGCT TTGCGCCAGG CTACATCACC ATGTTTATCG 2641 CTCGTCTGGT CATCGGCATG GGGATGGCGG GTGAATACGG TTCCAGCGCC ACCTATGTCA 2701 TTGAAAGCTG GCCAAAACAT CTGCGTAACA AAGCCAGTGG TTTTTTGATT TCAGGCTTCT 2761 CTGTGGGGGC CGTCGTTGCC GCTCAGGTCT ATAGCCTGGT GGTTCCGGTC TGGGGCTGGC 2821 GTGCGCTGTT CTTTATCGGC ATTTTGCCAA TCATCTTTGC TCTCTGGCTG CGTAAAAACA 2881 TCCCGGAAGC GGAAGACTGG AAAGAGAAAC ACGCAGGTAA AGCACCAGTA CGCACAATGG 2941 TGGATATTCT CTACCGTGGT GAACATCGCA TTGCCAATAT CGTAATGACA CTGGCGGCGG 3001 CTACTGCGCT GTGGTTCTGC TTCGCCGGTA ACCTGCAAAA TGCCGCGATC GTCGCTGTTC 3061 TTGGGCTGTT ATGCGCCGCA ATCTTTATCA GCTTTATGGT GCAGAGTGCA GGCAAACGCT 3121 GGCCAACGGG CGTAATGCTG ATGGTGGTCG TGTTGTTTGC TTTCCTCTAC TCATGGCCGA 3181 TTCAGGCGCT GCTGCCAACG TATCTGAAAA CCGATCTGGC TTATAACCCG CATACTGTAG 3241 CCAATGTGCT GTTCTTTAGT GGCTTTGGCG CGGCGGTGGG ATGCTGCGTA GGTGGCTTCC 3301 TCGGTGACTG GCTGGGAACC CGCAAAGCGT ACGTTTGTAG CCTGCTGGCC TCGCAGCTGC 3361 TGATTATTCC GGTATTTGCG ATTGGCGGCG CAAACGTCTG GGTGCTCGGT CTGTTACTGT 3421 TCTTCCAGCA AATGCTTGGA CAAGGGATCG CCGGGATCTT ACCAAAACTG ATTGGCGGTT 3481 ATTTCGATAC CGACCAGCGT GCAGCGGGCC TGGGCTTTAC CTACAACGTT GGCGCATTGG 3541 GCGGTGCACT GGCCCCAATC ATCGGCGCGT TGATCGCTCA ACGTCTGGAT CTGGGTACTG 3601 CGCTGGCATC GCTCTCGTTC AGTCTGACGT TCGTGGTGAT CCTGCTGATT GGGCTGGATA 3661 TGCCTTCTCG CGTTCAGCGT TGGTTGCGCC CGGAAGCGTT GCGTACTCAT GACGCTATCG 3721 ACGGTAAACC ATTCAGCGGT GCCGTGCCGT TTGGCAGCGC CAAAAACGAT TTAGTCAAAA 3781 CCAAAAGTTA ATCCTGTTGC CCGGTCTATG TACCGGGCCT TTCGCTAAGG GAAGATGTAT 3841 GTCGTTACTT GCACAACTGG ATCAAAAAAT CGCTGCTAAC GGTGGCCTGA TTGTCTCCTG 3901 CCAGCCGGTT CCGGACAGCC CGCTCGATAA ACCCGAAATC GTCGCCGCCA TGGCATTAGC 3961 GGCAGAACAG GCGGGCGCGG TTGCCATTCG CATTGAAGGT GTGGCAAATC TGCAAGCCAC 4021 GCGTGCGGTG GTGAGCGTGC CGATTATTGG AATTGTGAAA CGCGATCTGG AGGATTCTCC 4081 GGTACGCATC ACGGCCTATA TTGAAGATGT TGATGCGCTG GCGCAGGCGG GCGCGGACAT 4141 TATCGCCATT GACGGCACCG ACCGCCCGCG TCCGGTGCCT GTTGAAACGC TGCTGGCACG 4201 TATTCACCAT CACGGTTTAC TGGCGATGAC CGACTGCTCA ACGCCGGAAG ACGGCCTGGC 4261 ATGCCAAAAG CTGGGAGCCG AAATTATTGG CACTACGCTT TCTGGCTATA CCACGCCTGA 4321 AACGCCAGAA GAGCCGGATC TGGCGCTGGT GAAAACGTTG AGCGACGCCG GATGTCGGGT 4381 GATTGCCGAA GGGCGTTACA ACACGCCTGC TCAGGCGGCG GATGCGATGC GCCACGGCGC 4441 GTGGGCGGTG ACGGTCGGTT CTGCAATCAC GCGTCTTGAG CACATTTGTC AGTGGTACAA 4501 CACAGCGATG AAAAAGGCGG TGCTATGACC ACACTGGCGA TTGATATCGG CGGTACTAAA 4561 CTTGCCGCCG CGCTGATTGG CGCTGACGGG CAGATCCGCG ATCGTCGTGA ACTTCCTACG 4621 CCAGCCAGCC AGACACCAGA AGCCTTGCGT GATGCCTTAT CCGCATTAGT CTCTCCGTTG 4681 CAAGCTCATG CGCAGCGGGT TGCCATCGCT TCGACCGGGA TAATCCGTGA CGGCAGCTTG 4741 CTGGCGCTTA ATCCGCATAA TCTTGGTGGA TTGCTACACT TTCCGTTAGT CAAAACGCTG 4801 GAACAACTTA CCAATTTGCC GACCATTGCC ATTAACGACG CGCAGGCCGC AGCATGGGCG 4861 GAGTTTCAGG CGCTGGATGG CGATATAACC GATATGGTCT TTATCACCGT TTCCACCGGC 4921 GTTGGCGGCG GTGTAGTGAG CGGCTGCAAA CTGCTTACCG GCCCTGGCGG TCTGGCGGGG 4981 CATATCGGGC ATACGCTTGC CGATCCACAC GGCCCAGTCT GCGGCTGTGG ACGCACAGGT 5041 TGCGTGGAAG CGATTGCTTC TGGTCGCGGC ATTGCAGCGG CAGCGCAGGG GGAGTTGGCT 5101 GGCGCGGATG CGAAAACTAT TTTCACGCGC GCCGGGCAGG GTGACGAGCA GGCGCAGCAG 5161 CTGATTCACC GCTCCGCACG TACGCTTGCA AGGCTGATCG CTGATATTAA AGCCACAACT 5221 GATTGCCAGT GCGTGGTGGT CGGTGGCAGC GTTGGTCTGG CAGAAGGGTA TCTGGCGCTG 5281 GTGGAAACGT ATCTGGCGCA GGAGCCAGCG GCATTTCATG TTGATTTACT GGCGGCGCAT 5341 TACCGCCATG ATGCAGGTTT ACTTGGGGCT GCGCTGTTGG CCCAGGGAGA AAAATTATGA 5401 TGATGGGTGA AGTACAGTCA TTACCGTCTG CTGGGTTACA TCCTGCGTTA CAGGACGCGT 5461 TAACGCTGGC ATTAGCTGCC AGACCGCAAG AAAAAGCGCC GGGTCGTTAC GAATTACAGG 5521 GCGACAATAT CTTTATGAAT GTCATGACGT TTAACACTCA ATCGCCCGTC GAGAAAAAAG 5581 CGGAATTGCA CGAGCAATAC ATTGATATCC AGCTGTTATT AAACGGTGAG GAACGGATTC 5641 TGTTTGGCAT GGCAGGCACT GCGCGTCAGT GTGAAGAGTT CCACCATGAG GATGATTATC 5701 AGCTTTGCAG CACCATTGAT AACGAGCAAG CCATCATCTT AAAACCGGGA ATGTTCGCCG 5761 TGTTTATGCC AGGTGAACCG CATAAACCAG GATGCGTTGT CGGCGAGCCT GGAGAGATTA 5821 AAAAGGTTGT GGTGAAGGTT AAGGCTGATT TAATGGCTTA A (SEQ ID NO: 42) //

Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

1. A method for producing a sialylated oligosaccharide in a bacterium comprising: providing a bacterium, said bacterium comprising an exogenous sialyl-transferase, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, and a functional lactose permease gene, wherein said bacterium comprises an endogenous N-acetylneuraminate lyase gene (nanA) and an endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE) that are mutated and an endogenous N-acetylmannosamine kinase gene (nanK) that is not mutated; and culturing said bacterium in the presence of lactose.
 2. The method of claim 1, wherein said bacterium further comprises a mutation in endogenous N-acetylneuraminic acid transporter gene (nanT). 3.-5. (canceled)
 6. The method of claim 1, wherein the mutation comprises a null mutation.
 7. (canceled)
 8. The method of claim 1, wherein said sialic acid synthetic capability comprises an exogenous CMP-Neu5Ac synthetase gene (neuA), an exogenous sialic acid synthase gene (neuB), and an exogenous UDP-GlcNac 2-epimerase (neuC).
 9. The method of claim 1, wherein said exogenous sialyl-transferase gene is α(2,3) sialyl-transferase, α(2,6) sialyl-transferase, or α(2,8) sialyltransferase.
 10. The method of claim 1, wherein said sialylated oligosaccharide comprises 3′-sialyllactose (3′-SL) or 6′-sialyllactose (6′-SL).
 11. The method of claim 1, wherein said bacterium comprises a deleted or inactivated endogenous β-galactosidase gene.
 12. The method of claim 11, wherein said deleted or inactivated β-galactosidase gene comprises E. coli lacZ gene.
 13. The method of claim 1, wherein said bacterium comprises a recombinant β-galactosidase gene providing a low but detectable level of β-galactosidase activity.
 14. The method of claim 1, wherein said bacterium further comprises a deleted, inactivated, or mutated lacA gene.
 15. The method of claim 1, wherein said bacterium comprises an increased UDP-GlcNAc production capability.
 16. The method of claim 15, wherein said increased UDP-GlcNAc production capability comprises overexpression of (a) a nagC gene, a glmS gene, a glmY gene, a glmZ gene or any combination thereof; (b) an E. coli nagC gene; (c) a nagC gene and a glmS gene; (d) a nagC gene and a glmY gene; or (e) a nagC gene and a glmZ gene. 17.-20. (canceled)
 21. The method of claim 1, wherein said bacterium comprises E. coli.
 22. A method for producing an N-acetylglucosamine-containing oligosaccharide in a bacterium comprising: providing a bacterium, said bacterium comprising an exogenous UDP-GlcNAc:Galα/β-R 3-N-acetylglucosaminyltransferase gene and a functional lactose permease gene; and culturing said bacterium in the presence of lactose, wherein said bacterium comprises overexpression of (a) a nagC gene, a glmS gene, a glmY gene, a glmZ gene or any combination thereof; (b) a E. coli nagC gene; (c) a nagC gene and a glmS gene; (d) a nagC gene and a glmY gene; or (e) a nagC gene and a glmZ gene.
 23. The method of claim 22, wherein said bacterium comprises an increased UDP-GlcNAc production capability. 24.-28. (canceled)
 29. The method of claim 22, wherein said N-acetylglucosamine-containing oligosaccharide comprises any one selected from Lacto-N-triose 2 (LNT2), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lacto-N-fucopentaose V (LNF V), Lacto-N-difucohexaose I (LDFH I), Lacto-N-difucohexaose II (LDFH II), and Lacto-N-neodifucohexaose II (LFNnDFH II).
 30. The method of claim 22, wherein said bacterium is E. coli.
 31. A method of purifying a sialylated oligosaccharide produced by the method of claim 1, comprising binding said sialylated oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of said bacterium to a carbon column, and eluting said sialylated oligosaccharide from said column.
 32. A method of purifying an N-acetylglucosamine-containing oligosaccharide produced the method of claim 22, comprising binding said N-acetylglucosamine-containing oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of said bacterium to a carbon column, and eluting said sialylated oligosaccharide from said column. 33.-41. (canceled)
 42. The method of claim 1, further comprising retrieving said sialylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.
 43. The method of claim 22, further comprising retrieving said N-acetylglucosamine-containing oligosaccharide from said bacterium or from a culture supernatant of said bacterium. 